This document discusses research into the iron(II)- and 2-oxoglutarate-dependent halogenase SyrB2. The research aims to understand how SyrB2 positions its substrate for chlorination by incorporating cysteine residues into SyrB2 and its substrate carrier protein SyrB1. These cysteine variants will allow distance measurements using spin-labeling and electron magnetic resonance spectroscopy. Initial protein purification and expression testing of variants is being optimized. Once obtained, the variant proteins will help define how SyrB1 interacts with SyrB2 to precisely position the substrate for selective halogenation.
This document discusses machine learning approaches for protein fold class recognition when classes have low homology between amino acid sequences. It summarizes a dataset containing 420 protein domain structures divided into 51 fold classes that represent a difficult problem for protein structure prediction due to sequences within classes having less than 25% identity. The goal is to construct discriminant functions using machine learning methods to perform multi-class classification on this dataset and determine naturally compact subgroups of sequences and features.
Molecular and Structural Mechanism for Beta Barrel Proteins Incorporation in ...USTC, Hefei, PRC
Beta Barrel Proteins are important for membrane processes. This presentation is a simplified explanation of research article which elaborate incorporation of beta barrel proteins transport and incorporation and secretion snapshot from outer bacterial cell wall.
The document discusses the purification of a Sitag/RGD/His-tag fusion protein for use as a scaffold in tissue engineering. It examines different lysis and purification methods to extract the protein from E. coli cells. Lysing methods tested include freeze-thaw, detergents, and enzymes. Purification was initially attempted using nickel affinity chromatography via the His-tag, but residual proteins remained. Purification using silica and the silica-binding Sitag was then explored, but elution with L-lysine was ineffective. Increased washes and incubation improved elution, but some bacterial proteins still remained. Future work may use a silica gel system or alter purification conditions.
This document describes a study that used computational modeling to design novel inhibitors of BACE1, the enzyme involved in Alzheimer's disease pathogenesis. Researchers designed biaryl and fused-ring compounds targeting the S2 pocket of BACE1. They synthesized compounds based on these scaffolds and tested them for inhibitory activity against BACE1 and the related enzyme BACE2. One compound reduced amyloid beta 40 production in cells by 65%, indicating inhibitory activity against BACE1 with potential for treating Alzheimer's disease. The document discusses the rationale for targeting the flexible S2 pocket and for using less polar compounds that form fewer hydrogen bonds to gain selectivity against BACE2.
This document reviews the influence of nitric oxide (NO) on chloroplasts. It discusses how NO can directly interact with the photosynthetic apparatus by influencing photophosphorylation, electron transport, and the oxygen-evolving complex. It also reviews how NO can regulate gene expression in chloroplasts. The effects of NO on chloroplast function under physiological conditions like development and stress are discussed. Specifically, low levels of NO can stimulate plant growth and stress responses, while higher levels can inhibit photosynthesis.
This master's thesis examines the use of dual-focus fluorescence correlation spectroscopy (2f-FCS) to study the diffusion characteristics of fluorescently labelled sodium rhodaminylamino hyaluronate (Hya-Rh, 40 kDa) under various conditions, including the influence of concentration, physiological environment, alkali metal ions, and very low molecular weight hyaluronan. The document provides background on hyaluronic acid, single molecule detection techniques like FCS, and the theoretical basis and applications of the 2f-FCS method. The experimental work aims to evaluate and validate the use of 2f-FCS
This document summarizes Stephanie Reigh's master's thesis on the prevalence and utility of syn bases in functional RNA. It finds that while syn bases make up a small percentage of total bases, they often occur in functionally important locations in riboswitches, aptamers, and ribozymes. The thesis incorporates conformationally restricted nucleotides containing 8-bromopurines during transcription to stabilize native RNA structures and improve catalytic function. Future work will insert these nucleotides randomly to determine if they can enhance RNA activity.
The document describes a study that identified a novel mutation in the CLCNKB gene in a patient with an atypical mixed phenotype of Bartter-Gitelman syndrome. The researchers found a mutation (nt G499T, aa G167C) in the pore region of the ClC-Kb chloride channel protein. Functional studies showed the mutant channel had reduced cell surface expression and altered ion selectivity compared to the wild type. Kidney biopsies found the mutant protein localized incorrectly in the thick ascending limb and distal convoluted tubule cells. This novel mutation may contribute to the patient's unusual presentation of progressive chronic kidney disease.
This document discusses machine learning approaches for protein fold class recognition when classes have low homology between amino acid sequences. It summarizes a dataset containing 420 protein domain structures divided into 51 fold classes that represent a difficult problem for protein structure prediction due to sequences within classes having less than 25% identity. The goal is to construct discriminant functions using machine learning methods to perform multi-class classification on this dataset and determine naturally compact subgroups of sequences and features.
Molecular and Structural Mechanism for Beta Barrel Proteins Incorporation in ...USTC, Hefei, PRC
Beta Barrel Proteins are important for membrane processes. This presentation is a simplified explanation of research article which elaborate incorporation of beta barrel proteins transport and incorporation and secretion snapshot from outer bacterial cell wall.
The document discusses the purification of a Sitag/RGD/His-tag fusion protein for use as a scaffold in tissue engineering. It examines different lysis and purification methods to extract the protein from E. coli cells. Lysing methods tested include freeze-thaw, detergents, and enzymes. Purification was initially attempted using nickel affinity chromatography via the His-tag, but residual proteins remained. Purification using silica and the silica-binding Sitag was then explored, but elution with L-lysine was ineffective. Increased washes and incubation improved elution, but some bacterial proteins still remained. Future work may use a silica gel system or alter purification conditions.
This document describes a study that used computational modeling to design novel inhibitors of BACE1, the enzyme involved in Alzheimer's disease pathogenesis. Researchers designed biaryl and fused-ring compounds targeting the S2 pocket of BACE1. They synthesized compounds based on these scaffolds and tested them for inhibitory activity against BACE1 and the related enzyme BACE2. One compound reduced amyloid beta 40 production in cells by 65%, indicating inhibitory activity against BACE1 with potential for treating Alzheimer's disease. The document discusses the rationale for targeting the flexible S2 pocket and for using less polar compounds that form fewer hydrogen bonds to gain selectivity against BACE2.
This document reviews the influence of nitric oxide (NO) on chloroplasts. It discusses how NO can directly interact with the photosynthetic apparatus by influencing photophosphorylation, electron transport, and the oxygen-evolving complex. It also reviews how NO can regulate gene expression in chloroplasts. The effects of NO on chloroplast function under physiological conditions like development and stress are discussed. Specifically, low levels of NO can stimulate plant growth and stress responses, while higher levels can inhibit photosynthesis.
This master's thesis examines the use of dual-focus fluorescence correlation spectroscopy (2f-FCS) to study the diffusion characteristics of fluorescently labelled sodium rhodaminylamino hyaluronate (Hya-Rh, 40 kDa) under various conditions, including the influence of concentration, physiological environment, alkali metal ions, and very low molecular weight hyaluronan. The document provides background on hyaluronic acid, single molecule detection techniques like FCS, and the theoretical basis and applications of the 2f-FCS method. The experimental work aims to evaluate and validate the use of 2f-FCS
This document summarizes Stephanie Reigh's master's thesis on the prevalence and utility of syn bases in functional RNA. It finds that while syn bases make up a small percentage of total bases, they often occur in functionally important locations in riboswitches, aptamers, and ribozymes. The thesis incorporates conformationally restricted nucleotides containing 8-bromopurines during transcription to stabilize native RNA structures and improve catalytic function. Future work will insert these nucleotides randomly to determine if they can enhance RNA activity.
The document describes a study that identified a novel mutation in the CLCNKB gene in a patient with an atypical mixed phenotype of Bartter-Gitelman syndrome. The researchers found a mutation (nt G499T, aa G167C) in the pore region of the ClC-Kb chloride channel protein. Functional studies showed the mutant channel had reduced cell surface expression and altered ion selectivity compared to the wild type. Kidney biopsies found the mutant protein localized incorrectly in the thick ascending limb and distal convoluted tubule cells. This novel mutation may contribute to the patient's unusual presentation of progressive chronic kidney disease.
This document provides a summary of the education and experience of Prof. Dr Syeda Robina Gilani. It includes her positions, education history, research interests, publications, projects supervised, and other achievements. Some key details:
- She is currently the Chairperson of the Department of Chemistry at UET Lahore, Pakistan.
- Her education includes a Post Doc from Queen's University in 2008, a PhD from the University of Leicester in 1998, and an MSc from the University of Punjab in 1987-1989.
- Her research interests include environmental toxicology, coordination chemistry, functional food chemistry, and renewable energy.
- She has supervised several PhD, MPhil, and MSc the
Best possible natural ligands which were enlisted on NPACT website were screened ( aid of major drug likeness parameters - pkCSM) and docked with the 2OJG(Target protein) using autodock.
This document summarizes a thesis submitted by Gabriel Bendavit investigating the regulation of mTOR transcription by the transcription factor Nrf2. The introduction provides background on Nrf2 and its role in cellular defense responses. It describes previous research showing interactions between Nrf2 and the PI3K/AKT/mTOR signaling pathway. The hypothesis is that Nrf2 modulates mTOR expression. Experiments in lung cancer cell lines show that upregulating or downregulating Nrf2 correspondingly regulates mTOR protein levels. Luciferase assays and EMSA experiments indicate Nrf2 directly binds to and activates the mTOR promoter. Additional experiments suggest Nrf2 may also indirectly regulate other components of the PI3K pathway
The de novo designed hexameric coiled-coil protein CC-Hex was found to unfold with increasing pH when the leucine residue at position 24 was mutated to aspartate (L24D). Circular dichroism experiments showed the L24D hexamer was fully folded below pH 3 but less than 20% folded above pH 7.4. The models developed in this work aim to explain and predict this pH-dependent unfolding behavior of the L24D hexamer by considering the dominant physiochemical interactions affecting its stability as a function of pH.
Isolation of promoter and other regulatory elements.pptxShailendraPandey20
I apologize, I do not have enough context to answer questions about molecular biology concepts or experiments. My role is to summarize written documents.
This dissertation investigates luminescence properties of rare earth and transition metal ion doped fluoride crystals for applications in mercury-free fluorescent lamps. Single crystals of LiCaAlF6 doped with Tm3+ and Mn2+ were grown and their spectroscopic properties studied using synchrotron radiation and laser excitation. Fine structure was observed in emission spectra of Tm3+ due to crystal field splitting. Absorption bands associated with 3d → 4s transitions of Mn2+ were analyzed in various fluoride hosts. This work presents the first systematic analysis of Mn2+ in fluorides using synchrotron radiation.
Pharmacogenetics is the study of inherited genetic differences in drug metabolic pathways which can affect individual responses to drugs, both in terms of therapeutic effect as well as adverse effects. The term pharmacogenetics is often used interchangeably with the term pharmacogenomics which also investigates the role of acquired and inherited genetic differences in relation to drug response and drug behavior through a systematic examination of genes, gene products, and inter- and intra-individual variation in gene expression and function.
The International Journal of Engineering and Science (IJES)theijes
The International Journal of Engineering & Science is aimed at providing a platform for researchers, engineers, scientists, or educators to publish their original research results, to exchange new ideas, to disseminate information in innovative designs, engineering experiences and technological skills. It is also the Journal's objective to promote engineering and technology education. All papers submitted to the Journal will be blind peer-reviewed. Only original articles will be published.
This document is a summer training report submitted by Mahendra Kumar Sharma on biophysical studies of a βγ-crystallin homologue from the marine bacterium Hahella chejuensis. It describes purification and characterization of the recombinant protein. The introduction provides background on βγ-crystallin domains, their calcium binding motif, and objectives to determine the function of this domain within a multidomain protein from H. chejuensis. Methods used include affinity chromatography, ion exchange chromatography, and size exclusion chromatography to purify the protein for further biophysical analysis.
This document summarizes an experiment to engineer ascorbate peroxidase (APX) activity into cytochrome c peroxidase (CCP) by introducing the APX ascorbate-binding site into CCP. Specifically, the researchers replaced the ascorbate-binding loop and a critical arginine residue in CCP with the corresponding residues from APX to create a mutant called CCP2APX. While wild-type CCP showed no APX activity, CCP2APX was able to catalyze the peroxidation of ascorbate, demonstrating that the engineered ascorbate-binding site could bind ascorbate. Crystal structures of CCP2APX confirmed that the engineered binding site
The document summarizes the complete genome sequence analysis of Arthrobacter sp. PAMC25564, a bacterium isolated from cryoconite in Antarctica. Key findings include:
1) The genome is 4.17 million base pairs with a GC content of 66.74% and contains 3,829 genes.
2) Comparative genomic analysis with 16 other Arthrobacter strains identified genes involved in glycogen and trehalose metabolism pathways that may help it survive in cold environments.
3) The strain contains 108 genes encoding carbohydrate-active enzymes that could allow it to utilize polysaccharides and carbohydrates as an energy source in cold conditions.
This document summarizes a research team working on mathematical modeling and drug development for cancer treatment. The team is developing computational models of cancer processes and treatments to identify new chemotherapy compounds and improve treatment outcomes. Their work focuses on modeling microtubules and developing drugs that selectively target microtubule isoforms expressed in cancer cells. They use techniques like quantum mechanics, molecular dynamics simulations, and hybrid quantum/molecular mechanics methods to model drug-microtubule interactions and design new drugs. The overall goal is to leverage computational modeling to develop patient-specific cancer treatments.
This document describes a study characterizing the biosynthetic pathways of secondary metabolites in the filamentous fungus Aspergillus aculeatus. Gene deletion experiments using CRISPR-Cas9 and PCR-based gene targeting linked production of the hybrid polyketide-nonribosomal peptide acurin and the polyketide calbistrin A to two polyketide synthases. Bioinformatic analysis of the gene clusters predicted biosynthetic pathways for these compounds. A novel hybrid compound was also identified and linked to a putative hybrid polyketide synthase-nonribosomal peptide synthetase. The study expanded knowledge of fungal secondary metabolite biosynthesis.
This document describes the synthesis and characterization of a ruthenium polypyridyl complex. Key steps include:
1) Synthesizing a bipyridine ligand functionalized with a bromomethyl group and methyl substituent.
2) Attaching this ligand to diethyl malonate using a Mitsunobu reaction to form a chelating ligand.
3) Reacting this ligand with cis-bis(bipyridine)dichlororuthenium(II) dihydrate to form the target ruthenium complex.
4) Purifying and characterizing the complex using NMR spectroscopy and mass spectrometry. The complex exhibits photophysical properties suitable for applications
This document discusses enzymes and their applications. It begins by describing how enzymes are isolated from microorganisms and developed into strains for industrial use. It then discusses some key applications of enzymes in laundry detergents, where they help remove stains like blood, grease, and starch. Proteases target protein stains, lipases target fat stains, and amylases target starch stains. The document also briefly mentions other enzyme applications in industries like textiles and pulp/paper processing. It concludes with a short section on Hepatitis C virus.
This document describes research into developing small molecule "variators" that can reprogram the binding specificity of the BPTF protein reader domain. BPTF normally binds preferentially to trimethylated H3K4 histone marks, but the goal is to develop compounds that strengthen BPTF's interaction with monomethylated H3K4. This could potentially compensate for loss of the MLL2 methyltransferase in cancers. Through computational modeling and molecular dynamics simulations, the researchers identified probe molecules that stabilized a triple complex of BPTF, H3K4me1, and the probe. Virtual screening was then used to search databases for real molecules mimicking the optimal probes, with the aim of experimentally validating candidates
This document describes a new fluorimetric method for detecting and quantifying siderophores using Calcein Blue dye. Siderophores are iron-chelating compounds released by bacteria under iron-deficient conditions and can be used as markers for bacterial detection. The method exploits the property that Calcein Blue fluorescence is quenched by iron but regained when a stronger chelator like a siderophore removes the iron. Standard strains, clinical isolates, and media compositions were tested. A standard curve using the siderophore desferal allowed quantification of siderophores down to 50 nM. This sensitive, simple fluorescence-based method provides a new tool for bacterial detection within 7-8 hours.
Functionalizing Beta-Cyano Oligo(p-phenylene vinylene) Chromophores for use i...Alex Wink
This study synthesized and characterized a series of eight β-cyano oligo(p-phenylene vinylene) (cyano-OPV) molecules (dyes A-H) with different functional groups (hydrogen, methyl, dimethyl, cyano, fluoro, methoxy) placed on the terminal phenyl rings. The cyano-OPVs were synthesized via Knoevenagel condensation and characterized using NMR, TGA, DSC, UV-Vis spectroscopy, and photoluminescence measurements of the pure solids and solutions. Absorption properties, extinction coefficients, and emission wavelengths were determined to understand how changing the functional groups affects the molecules' ability to relax from an excited electronic
Phosphoproteomics of collagen receptor networks reveals SHP-2Maciej Luczynski
This document describes a study that used phosphoproteomics to analyze signaling networks downstream of collagen receptors. The researchers identified 424 phosphorylated proteins over seven time points after stimulating cells with collagen. Multiple clustering analysis revealed that phosphorylation sites on proteins like SHP-2, NCK1, LYN, and PIK3C2A strongly clustered with DDR2 phosphorylation dynamics, suggesting these proteins are candidate downstream effectors of DDR2 signaling. Biochemical validation showed SHP-2 tyrosine phosphorylation depends on DDR2 kinase activity. Targeted proteomics of lung cancer DDR2 mutants showed SHP-2 is phosphorylated by some mutants. This indicates SHP-2 is a key signaling node downstream of DDR
This document provides a summary of the education and experience of Prof. Dr Syeda Robina Gilani. It includes her positions, education history, research interests, publications, projects supervised, and other achievements. Some key details:
- She is currently the Chairperson of the Department of Chemistry at UET Lahore, Pakistan.
- Her education includes a Post Doc from Queen's University in 2008, a PhD from the University of Leicester in 1998, and an MSc from the University of Punjab in 1987-1989.
- Her research interests include environmental toxicology, coordination chemistry, functional food chemistry, and renewable energy.
- She has supervised several PhD, MPhil, and MSc the
Best possible natural ligands which were enlisted on NPACT website were screened ( aid of major drug likeness parameters - pkCSM) and docked with the 2OJG(Target protein) using autodock.
This document summarizes a thesis submitted by Gabriel Bendavit investigating the regulation of mTOR transcription by the transcription factor Nrf2. The introduction provides background on Nrf2 and its role in cellular defense responses. It describes previous research showing interactions between Nrf2 and the PI3K/AKT/mTOR signaling pathway. The hypothesis is that Nrf2 modulates mTOR expression. Experiments in lung cancer cell lines show that upregulating or downregulating Nrf2 correspondingly regulates mTOR protein levels. Luciferase assays and EMSA experiments indicate Nrf2 directly binds to and activates the mTOR promoter. Additional experiments suggest Nrf2 may also indirectly regulate other components of the PI3K pathway
The de novo designed hexameric coiled-coil protein CC-Hex was found to unfold with increasing pH when the leucine residue at position 24 was mutated to aspartate (L24D). Circular dichroism experiments showed the L24D hexamer was fully folded below pH 3 but less than 20% folded above pH 7.4. The models developed in this work aim to explain and predict this pH-dependent unfolding behavior of the L24D hexamer by considering the dominant physiochemical interactions affecting its stability as a function of pH.
Isolation of promoter and other regulatory elements.pptxShailendraPandey20
I apologize, I do not have enough context to answer questions about molecular biology concepts or experiments. My role is to summarize written documents.
This dissertation investigates luminescence properties of rare earth and transition metal ion doped fluoride crystals for applications in mercury-free fluorescent lamps. Single crystals of LiCaAlF6 doped with Tm3+ and Mn2+ were grown and their spectroscopic properties studied using synchrotron radiation and laser excitation. Fine structure was observed in emission spectra of Tm3+ due to crystal field splitting. Absorption bands associated with 3d → 4s transitions of Mn2+ were analyzed in various fluoride hosts. This work presents the first systematic analysis of Mn2+ in fluorides using synchrotron radiation.
Pharmacogenetics is the study of inherited genetic differences in drug metabolic pathways which can affect individual responses to drugs, both in terms of therapeutic effect as well as adverse effects. The term pharmacogenetics is often used interchangeably with the term pharmacogenomics which also investigates the role of acquired and inherited genetic differences in relation to drug response and drug behavior through a systematic examination of genes, gene products, and inter- and intra-individual variation in gene expression and function.
The International Journal of Engineering and Science (IJES)theijes
The International Journal of Engineering & Science is aimed at providing a platform for researchers, engineers, scientists, or educators to publish their original research results, to exchange new ideas, to disseminate information in innovative designs, engineering experiences and technological skills. It is also the Journal's objective to promote engineering and technology education. All papers submitted to the Journal will be blind peer-reviewed. Only original articles will be published.
This document is a summer training report submitted by Mahendra Kumar Sharma on biophysical studies of a βγ-crystallin homologue from the marine bacterium Hahella chejuensis. It describes purification and characterization of the recombinant protein. The introduction provides background on βγ-crystallin domains, their calcium binding motif, and objectives to determine the function of this domain within a multidomain protein from H. chejuensis. Methods used include affinity chromatography, ion exchange chromatography, and size exclusion chromatography to purify the protein for further biophysical analysis.
This document summarizes an experiment to engineer ascorbate peroxidase (APX) activity into cytochrome c peroxidase (CCP) by introducing the APX ascorbate-binding site into CCP. Specifically, the researchers replaced the ascorbate-binding loop and a critical arginine residue in CCP with the corresponding residues from APX to create a mutant called CCP2APX. While wild-type CCP showed no APX activity, CCP2APX was able to catalyze the peroxidation of ascorbate, demonstrating that the engineered ascorbate-binding site could bind ascorbate. Crystal structures of CCP2APX confirmed that the engineered binding site
The document summarizes the complete genome sequence analysis of Arthrobacter sp. PAMC25564, a bacterium isolated from cryoconite in Antarctica. Key findings include:
1) The genome is 4.17 million base pairs with a GC content of 66.74% and contains 3,829 genes.
2) Comparative genomic analysis with 16 other Arthrobacter strains identified genes involved in glycogen and trehalose metabolism pathways that may help it survive in cold environments.
3) The strain contains 108 genes encoding carbohydrate-active enzymes that could allow it to utilize polysaccharides and carbohydrates as an energy source in cold conditions.
This document summarizes a research team working on mathematical modeling and drug development for cancer treatment. The team is developing computational models of cancer processes and treatments to identify new chemotherapy compounds and improve treatment outcomes. Their work focuses on modeling microtubules and developing drugs that selectively target microtubule isoforms expressed in cancer cells. They use techniques like quantum mechanics, molecular dynamics simulations, and hybrid quantum/molecular mechanics methods to model drug-microtubule interactions and design new drugs. The overall goal is to leverage computational modeling to develop patient-specific cancer treatments.
This document describes a study characterizing the biosynthetic pathways of secondary metabolites in the filamentous fungus Aspergillus aculeatus. Gene deletion experiments using CRISPR-Cas9 and PCR-based gene targeting linked production of the hybrid polyketide-nonribosomal peptide acurin and the polyketide calbistrin A to two polyketide synthases. Bioinformatic analysis of the gene clusters predicted biosynthetic pathways for these compounds. A novel hybrid compound was also identified and linked to a putative hybrid polyketide synthase-nonribosomal peptide synthetase. The study expanded knowledge of fungal secondary metabolite biosynthesis.
This document describes the synthesis and characterization of a ruthenium polypyridyl complex. Key steps include:
1) Synthesizing a bipyridine ligand functionalized with a bromomethyl group and methyl substituent.
2) Attaching this ligand to diethyl malonate using a Mitsunobu reaction to form a chelating ligand.
3) Reacting this ligand with cis-bis(bipyridine)dichlororuthenium(II) dihydrate to form the target ruthenium complex.
4) Purifying and characterizing the complex using NMR spectroscopy and mass spectrometry. The complex exhibits photophysical properties suitable for applications
This document discusses enzymes and their applications. It begins by describing how enzymes are isolated from microorganisms and developed into strains for industrial use. It then discusses some key applications of enzymes in laundry detergents, where they help remove stains like blood, grease, and starch. Proteases target protein stains, lipases target fat stains, and amylases target starch stains. The document also briefly mentions other enzyme applications in industries like textiles and pulp/paper processing. It concludes with a short section on Hepatitis C virus.
This document describes research into developing small molecule "variators" that can reprogram the binding specificity of the BPTF protein reader domain. BPTF normally binds preferentially to trimethylated H3K4 histone marks, but the goal is to develop compounds that strengthen BPTF's interaction with monomethylated H3K4. This could potentially compensate for loss of the MLL2 methyltransferase in cancers. Through computational modeling and molecular dynamics simulations, the researchers identified probe molecules that stabilized a triple complex of BPTF, H3K4me1, and the probe. Virtual screening was then used to search databases for real molecules mimicking the optimal probes, with the aim of experimentally validating candidates
This document describes a new fluorimetric method for detecting and quantifying siderophores using Calcein Blue dye. Siderophores are iron-chelating compounds released by bacteria under iron-deficient conditions and can be used as markers for bacterial detection. The method exploits the property that Calcein Blue fluorescence is quenched by iron but regained when a stronger chelator like a siderophore removes the iron. Standard strains, clinical isolates, and media compositions were tested. A standard curve using the siderophore desferal allowed quantification of siderophores down to 50 nM. This sensitive, simple fluorescence-based method provides a new tool for bacterial detection within 7-8 hours.
Functionalizing Beta-Cyano Oligo(p-phenylene vinylene) Chromophores for use i...Alex Wink
This study synthesized and characterized a series of eight β-cyano oligo(p-phenylene vinylene) (cyano-OPV) molecules (dyes A-H) with different functional groups (hydrogen, methyl, dimethyl, cyano, fluoro, methoxy) placed on the terminal phenyl rings. The cyano-OPVs were synthesized via Knoevenagel condensation and characterized using NMR, TGA, DSC, UV-Vis spectroscopy, and photoluminescence measurements of the pure solids and solutions. Absorption properties, extinction coefficients, and emission wavelengths were determined to understand how changing the functional groups affects the molecules' ability to relax from an excited electronic
Phosphoproteomics of collagen receptor networks reveals SHP-2Maciej Luczynski
This document describes a study that used phosphoproteomics to analyze signaling networks downstream of collagen receptors. The researchers identified 424 phosphorylated proteins over seven time points after stimulating cells with collagen. Multiple clustering analysis revealed that phosphorylation sites on proteins like SHP-2, NCK1, LYN, and PIK3C2A strongly clustered with DDR2 phosphorylation dynamics, suggesting these proteins are candidate downstream effectors of DDR2 signaling. Biochemical validation showed SHP-2 tyrosine phosphorylation depends on DDR2 kinase activity. Targeted proteomics of lung cancer DDR2 mutants showed SHP-2 is phosphorylated by some mutants. This indicates SHP-2 is a key signaling node downstream of DDR
Phosphoproteomics of collagen receptor networks reveals SHP-2
Final Approved Thesis
1. THE PENNSYLVANIA STATE UNIVERSITY
SCHREYER HONORS COLLEGE
EBERLY COLLEGE OF SCIENCE
WHAT TO EXPECT WHEN EXPRESSING: CYSTEINE-CONTAINING SYRB2
VARIANT EXPRESSION TESTS IN PREPARATION FOR SPIN-LABELING
EXPERIMENTS
ZACHARY ANDREW SPRINGER
FALL 2016
A thesis
submitted in partial fulfillment of
the requirements
for a baccalaureate degree
in Science
with honors in Science
Reviewed and approved* by the following:
J. Martin Bollinger, Jr.
Professor of Chemistry and Biochemistry and Molecular Biology
Thesis Supervisor
Alicia Kehn
Advising Program Coordinator, Science and Premedicine; BS/MBA Advisor
Honors Adviser
* Signatures are on file in the Schreyer Honors College.
2. i
Abstract
Iron(II)- and 2-(oxo)glutarate-dependent (Fe/2OG) oxygenases are a versatile and
exciting class of enzymes that have unusual properties and help synthetize important natural
products. The members of this class known as halogenases can append chlorine or bromine to
even completely unactivated aliphatic carbon centers. Syringomycin Biosynthesis Enzyme 2
(SyrB2) is one such halogenase found in Pseudomonas syringae. SyrB2 catalyzes the
chlorination of its native substrate, L-threonine (Thr), to form 4-chloro-L-threonine [1]. SyrB2
requires that the Thr target be delivered by the substrate or "carrier protein," SyrB1, which has a
covalently linked phosphopantetheine arm that carries the amino acid by a thioester linkage. Thr
chlorination affords a precursor to the antifungal and phytotoxic lipodepsipeptide, syringomycin
E. The biological activity of this compound depends on the presence of the halogen [2]. SyrB2
has been characterized by crystallography, and its active site has an Fe(II) center with octahedral
coordination to chloride, 2OG, histidine residues 116 and 235, and water [3]. Recent studies have
suggested that an important means by which the enzyme directs its chlorination reaction is
control of the disposition of the Thr C4 methyl group relative to the cofactor, but the structure of
the halogenase•aminoacyl-carrier-protein complex has not yet been reported. Consequently, the
manner in which the phosphopantetheine arm threads into the active site to position the substrate
is unknown. To test hypotheses regarding the trajectory of approach and where on the halogenase
the phosphopantetheine arm threads into the active site, we incorporated cysteine residues at
strategic positions in the SyrB2 and SyrB1 proteins to permit thiol-directed spin-labeling and
measurement of distance restraints by advanced electron magnetic resonance spectroscopy.
Expression and purification of these variants is currently being optimized. Once obtained and
successfully spin-labeled, the variant proteins will permit the nature of the SyrB1-SyrB2
interaction that precisely positions the Thr-methyl target for selective halogenation to be defined.
3. ii
TABLE OF CONTENTS
List of Figures..........................................................................................................................iv
List of Tables ...........................................................................................................................vi
Abstract....................................................................................................................................i
Acknowledgements..................................................................................................................vi
Introduction..............................................................................................................................1
Chapter 1.1. Proteins, Enzymes, and Metalloenzymes ....................................................1
Chapter 1.2. Halogenating Metalloenzymes ....................................................................2
Chapter 1.3. Fe/2OG Enzymes and the Facial Triad........................................................3
Chapter 1.4. Fe/2OG Halogenases...................................................................................6
Chapter 1.4.a. Fe/2OG Halogenases: Introduction ..........................................................6
Chapter 1.4.b. Fe/2OG Halogenases: Cytotrienin Halogenase (CytC3) ..........................8
Chapter 1.4.c. Fe/2OG Dependent Halogenases: Syringomycin Biosynthesis
Enzyme 2 (SyrB2)....................................................................................................9
Chapter 1.4.d. Fe/2OG Halogenases: SyrB2 Active Site Structure and Remaining
Questions..................................................................................................................11
Materials and Methods.............................................................................................................14
Chapter 2.1 Materials.......................................................................................................14
Chapter 2.2. Polymerase Chain Reaction (PCR) Mutagenesis ........................................16
Chapter 2.2.a. PCR Reaction Mix....................................................................................16
Chapter 2.2.b. PCR Protocol............................................................................................17
Chapter 2.2.c. PCR Modifications ...................................................................................19
Chapter 2.3. Agarose Gel Electrophoresis .......................................................................21
Chapter 2.4. Phosphorylation/Ligation (KL) Reaction....................................................22
Chapter 2.5. Transformation of the Ligated PCR Product...............................................23
Chapter 2.6. DNA Extraction...........................................................................................24
Chapter 2.6.a. Initial DNA Extraction and Concentration ...............................................24
Chapter 2.6.b. Modifications to DNA Extraction Protocol..............................................25
Chapter 2.7 Sequencing ...................................................................................................26
Chapter 2.8. Preparation of New BL21 Competent Cells................................................28
Chapter 2.9. Protein Growth ............................................................................................29
Chapter 2.10. Purification ................................................................................................30
Chapter 2.10.a. Initial Purification Procedure..................................................................30
4. iii
Chapter 2.10.b. Modified Protein Expression and Purification through Expression
Tests .........................................................................................................................31
Chapter 2.11. SDS-PAGE Electrophoresis ......................................................................36
Chapter 2.12. Dialysis......................................................................................................38
Chapter 2.13. UV/VIS Spectroscopy and Protein Concentration ....................................39
Results ....................................................................................................................................43
Chapter 3.1 Cysteine Removal and Cysteine Addition Process.......................................43
Chapter 3.2 Results of Initial Protein Purifications and UV/VIS Absorption Analysis...44
Chapter 3.3 Expression Test Results................................................................................48
Discussion and Direction of Future Research..........................................................................57
Chapter 4.1 "Around-the-Horn" Mutagenesis..................................................................57
Chapter 4.2 Initial SyrB2 Protein Expression and UV/VIS Result Analysis...................58
Chapter 4.3 Expression Test Discussion..........................................................................59
Chapter 4.4 Future Direction of Research........................................................................61
Literature Cited........................................................................................................................63
Reference Documents: Appendix and CV...............................................................................69
Appendix A......................................................................................................................69
5. iv
LIST OF FIGURES
Figure 1-1. TauD Mechanism..................................................................................................3
Figure 1-2. Hypothesis for the Mechanism of the Hydroxylation Reactions in Fe/2OG
Enzymes...........................................................................................................................5
Figure 1-3. Proposed mechanism for the Fe/2OG halogenase, CytC3. ...................................8
Figure 1-4. Modeling of the SyrB2 Crystal Structure..............................................................10
Figure 1-5. Possible Geometries of O2 Addition During the Chlorination Reaction of
SyrB2. ..............................................................................................................................12
Figure 2-1. DNA Sequencing Request Form...........................................................................27
Figure 3-1. Ethidium Bromide Stained Agarose Gel of PCR Reactions Visualized under
UV Light ..........................................................................................................................44
Figure 3-2. SDS-PAGE Gel after Initial SyrB2 S258C Purification .......................................45
Figure 3-3. UV/VIS Results for SyrB2 S258C........................................................................46
Figure 3-4. SDS PAGE Gel Showing SyrB2 Q245C Variant Protein in Loading and
Column-wash Eluates (High Contrast) ............................................................................47
Figure 3-5. Expression Test 1 Gel (High Contrast) .................................................................49
Figure 3-6. Expression Test 2 Gel Result ................................................................................51
Figure 3-7. 6 mg IPTG Induced, SyrB2 S258C Expressing E. Coli Pellet and Purification
Gel....................................................................................................................................53
Figure 3-8. Expression Test 3 Gel ...........................................................................................55
Figure 3-9. Purification of SyrB2 S258C from Third Expression Test ...................................56
6. v
LIST OF TABLES
Table 2-1. DNA Primer Sequences Used in "Around-the Horn" Mutagenesis of SyrB2........15
Table 2-2. DNA Primer Sequences Used in "Around-the-Horn" Mutagenesis for SyrB1 ......16
Table 2-3. Mix for “Around-the-horn” site-directed mutagenesis via PCR ............................17
Table 2-4. PCR steps for Bio-Rad C1000 Thermal Cycler......................................................18
Table 2-5. Modified PCR Mix.................................................................................................19
Table 2-6. Annealing Temperature Gradient with Markers for Samples.................................20
Table 2-7. KL Reaction Mix....................................................................................................22
Table 2-8. Modifications to DNA Elution Procedure..............................................................26
Table 2-9. Variables and Control Values in Expression Test Trials........................................32
Table 2-10. Absorbance and IPTG Amount for First Expression Test....................................34
Table 2-11. Modified Equilibration and Elution Buffers.........................................................35
Table 2-12. IPTG Concentrations and Time of Incubation after Induction in the Third
Expression Test................................................................................................................36
Table 2-13. SDS-PAGE Acrylamide Gel Recipe ....................................................................38
Table 2-14. UV/VIS Spectroscopy Settings ............................................................................40
Table 2-15. Beer’s Law Equation ............................................................................................41
Table 3-1. Expression Test Gel 1 Key.....................................................................................48
Table 3-2. Expression Test 2 Gel Key.....................................................................................50
Table 3-3. Expression Test 3 Gel Key.....................................................................................54
7. vi
Acknowledgements
First of all, I would like to thank my family and friends for their support of me
throughout this research process. I could not have done it without the guidance, advice, and
understanding. Additionally, I must also thank everyone who convinced me to leave lab and go
home sometimes too. I would like to give a special thanks to Alicia Kehn, Henriette Evans, and
the Science BS/MBA program for believing in me. I cannot wait to start the next chapter of my
Penn State career and have this program to thank for helping me to find the best path for me.
I would next like to thank Dr. Bollinger and Dr. Krebs for supporting my research and for
providing me with this research position. It was a privilege and an honor to work in your lab and I
am very thankful for this opportunity. I would like to say a very special thanks to Ryan Martinie
for his support throughout the researching and writing process. I put hundreds of hours of work
into this project, and I could not have done it without his help, guidance, and friendship. I would
also like to thank Juan, Chris, Beth, Wei-Chen, Susan, Bo, Jovan, and all of the members of the
Bollinger-Krebs Group who I got to know because of this research. I will miss Waffle Fridays,
lab jokes, and lots of sarcasm. Thanks for making this experience so enjoyable. I have grown
greatly as a student, as a professional, and as a person because of this lab. I learned that despite
the best of efforts, a research project can be unexpected. It can be frustrating, and little mistakes
can make a large difference. In these failures however, great life lessons can be learned about
ones’ self that transcend the laboratory setting. On a smaller scale, I learned that I could have a
successful research project without meeting my end goal. For these lessons, I am forever thankful
to this lab and for the research project that I had the privilege to work on.
8. 1
Chapter 1 Introduction
Chapter 1.1. Proteins, Enzymes, and Metalloenzymes
Proteins are biological polymers of amino acids that play critical but varied roles within
the cells of organisms. Proteins serve as structural components, as channels for small molecules,
as internal and external signals for cells, and as catalysts of biochemical reactions, among many
other roles. All proteins that catalyze reactions are called enzymes. They lower the activation
energy required for crucial biochemical processes that would be too slow in their absence.
Enzyme reactions take place within their active sites, the region where the substrate, or reactant
molecule that will go through the enzyme-mediated reaction, binds to the enzyme.
Metalloenzymes are a particularly interesting group of enzymes that contain one or more
metal ions at their active sites. At least half of all proteins are thought to contain metals. [6] Metal
cofactors such as iron, manganese, copper, zinc, and magnesium are commonly found in the
active centers of metalloenzymes. These metal ions can be coordinated to a number of different
amino acids and molecules that may participate in enzymatic reactions or assist in the structural
integrity of the enzyme. Metals are important to enzyme function because they can be used to
catalyze difficult reactions. Metals can activate unreactive molecules, such as inert H2, N2, carbon
monoxide, and methane. [7] Metals that catalyze the activation of unactivated carbon centers are
of interest to scientists, in part because of the possibilities these enzymes pose in creating natural
products that may be used in medicinal, industrial, or other applications. Reactions can happen at
one single, intrinsically unreactive carbon with remarkable effectiveness. Metalloenzymes create
products with accurate stereochemistry and regiochemistry, even when performing a reaction
hundreds or thousands of times. These products are usually more beneficial than racemic mixtures
9. 2
(products with mixed stereochemistry) because, in many cases, stereoisomers may have
completely different functions, and oftentimes the "wrong" isomer may even have a negative
effect on the activity of the "correct" version of a molecule. The effectiveness of biological
reactions eliminates many of the problems (e.g., lack of stereochemical accuracy, toxicity of
reagents) scientists face with simpler chemical reagents and catalysts. [8]
Chapter 1.2. Halogenating Metalloenzymes
Several classes of metalloenzymes add halogens to unactivated carbons on their substrate
molecules. These halogenated products serve as hormones, growth regulators, antibiotics, or
toxins. [9,10] Several major kinds of metalloenzyme halogenases exist, and they can be classified
by the metal within their active site as well as by what additional molecules must be bound to this
metal to allow for halogenation. Classes of halogenases that have been studied recently include
haloperoxidases, flavin-dependent halogenases, and iron(II)- and 2-(oxo)glutarate-dependent
(Fe/2OG) halogenases. Haloperoxidases may have either a vanadium metal center or a heme-iron
center and create a hypohalous acid that halogenates an sp2
-hyrbidized carbon. Flavin-dependent
halogenation uses the same hypohalous-acid-based reaction mechanism. This kind of
halogenation was first studied in an enzyme called RebH, which chlorinates a tryptophan at
carbon C7 to provide a precursor to the natural product, rebaccamycin. The reaction is thought to
proceed by a mechanism in which FADH2 reacts with O2 to make a C4a-peroxy-flavin
intermediate. Chloride attacks this oxidizing intermediate, forming hypochlorous acid, HOCl,
which chlorinates the amine side chain of a lysine residue. The lysine-chloramine intermediate
then regioselectively chlorinates the tryptophan substrate. [11]
10. 3
Chapter 1.3. Fe/2OG Enzymes and the Facial Triad
The non-heme-iron(II) enzyme class has many important members that must be discussed
before the halogenase subclass is specifically considered. Taurine:2-(oxo)glutarate dioxygenase
(TauD) is a well-studied Fe/2OG enzyme that has been studied extensively and has provided a
great deal of information about the class. TauD is a critical enzyme for microbes, allowing them
to grow in aerobic environments that contain little cysteine or sulfate. This enzyme hydroxylates
taurine (Figure 1-1). In this reaction, 2OG, initially coordinated in bidentate mode via its C1
carboxylate and C2 carbonyl, is converted to succinate and gives off CO2 (Figure 1-2.a.). The
reaction is initiated when O2 adds to the Fe(II) cofactor, yielding a Fe(III)-superoxide complex.
As shown by freeze-quench Mӧssbauer experiments, an Fe(IV)-oxo (ferryl) intermediate then
forms. This complex is the functional hallmark of non-heme-iron(II) enzymes in general and
Fe/2OG enzymes specifically. [12] The highly electrophilic oxo group can abstract a hydrogen
atom (H•) from C1 of the substrate taurine, leaving a radical on the substrate and a hydroxyl
group coordinated to the iron [now Fe(III)]. At this point, the hydroxyl group is attacked by the
radical on the substrate, giving the final hydroxylated product. [13, 14]
Figure 1-1. TauD Mechanism
11. 4
The figure above (from Ye et al., JACS 2010 [13]) shows the TauD mechanism that converts
taurine to 1-hydroxytaurine. This product breaks down in solution to sulfonate and
aminoacetylaldehyde. The sulfonate can then be used as a source of sulfur for many biological
functions. Formation of the Fe(III)-superoxide complex (I), 2OG decarboxylation to form the
ferryl intermediate (III), and H• abstraction (to form IV) are common steps in the mechanisms of
Fe/2OG and related non-heme-iron enzymes. [13]
In TauD and related enzymes, [as pictured in (Figure 1-1) and (Figure 1-2.b.)] the
positions of the histidine and aspartic acid residues relate to the functionality of the enzyme and
are conserved in most non-heme-iron(II) oxygenases. This active site structure containing an
iron(II) ion coordinated by two histidine residues and a carboxylate group (from the amino acid
aspartic acid or glutamic acid) is known as the "facial triad" structural motif. [4] Most non-heme
iron(II) oxygenases and oxidases, both the Fe/2OG and 2OG-independent members, have this
facial triad structure. These enzymes form ferryl intermediates. Generally, substitution of the
histidines or aspartic/glutamatic acid residues eliminates enzymatic activity (with a few
exceptions). [15,16] A study reported in 2006 concluded that a consensus mechanism exists for
the Fe/2OG oxygenase family (Figure 1-2.b.). [17] This research was successful in trapping the
high-spin (S = 2) Fe(IV) complex and the post-hydroxylation, succinate-coordinated, high-spin
Fe(II) product state [J and V in (Figure 1-2b.)] within the Fe/2OG prolyl-4-hydroxylase from
Paramecium bursaria Chlorella virus 1 (PBCV1 P4H). The high-spin Fe(IV) intermediate state,
which was previously characterized in TauD, was especially critical in confirming that the
hydroxylation mechanism is conserved within this family. The high-spin Fe(IV) and Fe(II) states
were characterized by stopped-flow absorption and freeze-quench Mӧssbauer spectroscopies and
were shown to be analogous to the H•-abstracting complex in TauD. The Fe(IV) state was also
12. 5
shown to exhibit large a substrate deuterium kinetic isotope effect on its decay, as was also seen
in TauD. This mechanism is thought to be additionally conserved in other enzymes, such as
sulfate:αKG dioxygenase (AtsK). [18]
(a.) (b.)
Figure 1-2. Hypothesis for the Mechanism of the Hydroxylation Reactions in Fe/2OG Enzymes
(a.) The figure above (from Hegg et al. European Journal of Biochemistry 1997 [4]) shows the
reaction catalyzed by the facial triad containing, Fe/2-(oxo)acid oxygenases. (b.) The figure to the
right (from Hoffart et al. PNAS 2006 [17]) shows the consensus Fe/2OG mechanism for the
specific case of prolyl-4-hydroxylase. Intermediate states J and V were detected by both stopped-
flow (SF) absorption and freeze-quench (FQ) Mӧssbauer experiments in P4H and TauD.
The conserved nature of these enzymes has allowed researchers to formulate specific
hypotheses as to what differences are important in specifying the divergent outcomes of family
members. Diketone-cleaving dioxygenase 1 (DKE1) is an non-heme-iron enzyme that has a 3-
histidine triad. Inherently, the imidazole ligand from the extra histidine imposes a stronger ligand
13. 6
field than a carboxylate. The His3 triad is also a less effective in electron donation because it leads
to a more positively charged first coordination sphere. [19] In comparison with the typical
Fe/2OG model, in which the acid is decarboxylated via C1-C2 cleavage, the His3 model creates
entirely different products by cleaving C2-C3, adjacent to rather than between the diketone
moiety. This research demonstrates the importance of the structure of the ligand sphere in
directing the enzyme reaction’s outcome. [20]
The conserved structure of the “facial triad” is remarkable in its uniformity throughout
the Fe/2OG oxygenases. However, the usefulness of this structure in predicting the function and
mechanism of enzymes is highlighted by the different reaction outcomes that occur because of
variations in this amino acid coordination motif. Modifications to the “facial triad” structure
allow the Fe/2OG halogenases to catalyze an entirely different reaction, and this enzyme class
will be examined in the next section.
Chapter 1.4.a. Fe/2OG Halogenases: Introduction
The structure of the facial triad and relation to function is particularly important to the
Fe/2OG halogenases, as first exemplified by studies on CytC3 and Syringomycin Biosynthesis
Enzyme 2 (SyrB2). These halogenation enzymes contain histidines in accordance with the facial
triad structure. As expected from the oxygenase model, the iron cofactor is also associated with
2OG (bidentate coordination) and reacts with molecular oxygen. However, the cofactors in these
enzymes are coordinated by a halide ion in the place normally occupied by the carboxylate group.
An alanine or glycine replaces the aspartate/glutamate amino acid in the sequences of these
halogenases, including CytC3 and SyrB2. The smaller size and absence of a coordinating
functional group in the alanine creates space for chloride to coordinate directly to the iron. [21]
14. 7
Mechanistically, Fe/2OG halogenases are very similar to their dioxygenase counterparts.
2OG is decarboxylated, the hallmark ferryl intermediate is formed, and a hydrogen atom is
abstracted by the ferryl species. As pictured in Figures 1 and 2 (b.), the next step in the
hydroxylase mechanism is coupling of the substrate radical with the bound hydroxo ligand to the
Fe(III) cofactor state. The halogenase outcome differs in that, after H• abstraction, the chloro
ligand of the cognate Fe(III) complex, rather than hydroxo, couples with the substrate radical.
[21] The radical coupling steps involving the hydroxo and chloro ligands are expected to have
similar barrier heights [22]. Understanding how the halogenases direct chlorination, then,
became a central focus of research on the halogenases.
The structural and mechanistic similarities between the halogenase and hydroxylase
(dioxygenase) enzymes have led scientists to attempt reciprocally re-engineer them for the
opposite outcome. The alanine has been replaced in the halogenase model with aspartic or
glutamic acid via mutagenesis to form the facial triad scheme, and the reverse has been done in
hydroxylases. In neither case was that single substitution sufficient to reverse the outcome, and, in
general, the variant proteins have generally proven to be inefficient catalysts, at best. [21] Amino
acid sequence and structure of the first coordination sphere do not seem to be the sole
determinants of the functionality of the enzyme, but, as has been shown in both hydroxylases and
halogenases, are nonetheless important for control. [15, 21] Past work from my research group
showed that substrate positioning is a major contributor to reaction chemoselectivity in the
halogenases. [5] This topic will be elaborated upon in the next two sections discussing the
characterized Fe/2OG halogenase, CytC3 and SyrB2 (the latter being the focus of my thesis
work).
15. 8
Chapter 1.4.b. Fe/2OG Halogenases: Cytotrienin Halogenase (CytC3)
CytC3 is one of the first Fe/2OG halogenases discovered. It performs two sequential
chlorination reactions on the same carbon of its substrate. The final product is γ,γ-dichloro-L-2-
aminobutyrate, a natural antibiotic produced by Streptomyces bacteria found in soil. The substrate
of CytC3, L-2-aminobutyric acid (Aba), is linked as a thioester to the phosphopantetheine arm of
CytC2, a stand-alone thiolation or carrier protein domain, and is delivered to the active site of
CytC3 by CytC2. As discussed in the previous section, CytC3 has chloride, two histidine
residues, 2OG, and water coordinated to its active site in the O2-reactive complex. The working
hypothesis for the mechanism of the reaction is shown in Figure 1-3. [21, 23]
Figure 1-3. Proposed mechanism for the Fe/2OG halogenase, CytC3.
(Adapted from Galoniḉ et al. Nature Chemical Biology 2007 [23]).
16. 9
No crystal structure of CytC3 containing a chloride ligand has yet been reported.
Nonetheless, the available structure (that is, the structure without a chloride ligand) gives several
important clues to the importance of ligand and native amino acid positions to allow for
enzymatic activity. Researchers determined that the open active site structure was missing
structural components that would allow for chloride binding. For example, the open crystal
structure of CytC3 lacked a hydrogen-bond network and a hydrophobic space for chloride
coordination that are both present in the crystallized form of another halogenase of interest,
SyrB2. [21]
Chapter 1.4.c. Fe/2OG Dependent Halogenases: Syringomycin Biosynthesis Enzyme 2
(SyrB2)
SyrB2 is a halogenase very closely related to CytC3. In fact, the two enzymes are 58%
identical and contain 71% similar residues. Additionally, SyrB2 is thought to have chloride
binding loops and a hydrophobic pocket similar to those in CytC3 [21]. SyrB2, found in
Pseudomonas syringae, helps create the antifungal phytotoxin syringomycin by chlorinating the
amino acid threonine to create 4-Cl-L-Thr. This chlorination step is critical, as without the
chlorine, syringomycin is four times less potent [21]. The 66 kDa carrier protein, SyrB1, delivers
the L-Thr chlorination target to the SyrB2 active site via the phosphopantetheine arm, as in the
CytC2/CytC3 system.
SyrB2 has served as the model Fe/2OG halogenase for several reasons: i) it was one of
the first of this class to have been discovered, ii) the crystal structure, including the iron cofactor,
was the first to have been solved (Figure 1-4), iii) the iron (IV)-oxo intermediate was
characterized by Mössbauer and Extended X-ray Absorption Fine Structure (EXAFS)
spectroscopies [24]. The discovery and subsequent research on SyrB2 has challenged what is
17. 10
known about Fe/2OG dependent enzymes and continues to be central to our understanding of
reaction outcome.
Figure 1-4. Modeling of the SyrB2 Crystal Structure
The figures above (from Blasiak et al. Nature 2006 [3]) give various models of the crystal
structure of SyrB2. The brown sphere represents the iron cofactor, around which the chloride
(green), 2OG, histidines (116 and 235), and water (blue) are coordinated. The water molecule is
thought to be displaced upon binding of the aminoacyl-SyrB1 substrate prior to the reaction with
O2. A change in coordination positions may occur to accommodate the formation of the Fe(III)-
superoxide intermediate state or in later steps. [25, 5]
18. 11
Chapter 1.4.d. Fe/2OG Halogenases: SyrB2 Active Site Structure and Remaining
Questions
As was mentioned earlier, attempts to alter the active site structure of SyrB2 to mimic
that of Fe/2OG hydroxylases not only failed to induce efficient hydroxylation of threonine but
also eliminated halogenation activity. [21] However, SyrB2 will actually perform hydroxylation
of certain non-native amino acid substrates. The SyrB1 phosphopantetheine arm was used to
deliver either L-2-aminobutyricAcid (Aba) or L-norvaline (Nva) instead of L-Thr. SyrB2
catalyzes both chlorination and hydroxylation of Aba at C4 (the same carbon activated in Thr) and
almost exclusively hydroxylatesNva at C5. [5] This study determined that the rate of H•
abstraction correlated with the fraction of the hydroxylation outcome. As the H•-abstraction rate
increases, so did the proficiency of •OH radical transfer. Therefore, it was proposed that H•
abstraction is slow as a consequence of structural adaptations to enforce chlorination of the native
substrate. It was further suggested that the nature of the adaptation lies in how the enzyme
positions its substrate, and that the success of the subsequent chlorination reaction depends on this
unusual positioning. Threonine was suggested to be positioned so that the C4 carbon, which
donates the H• to the ferryl complex, is situated away from the oxo ligand and closer to chlorine.
In fact, later EPR spectroscopic analysis provided evidence that the C4-H bond might even be
nearly perpendicular to the oxo ligand. In this study, NO was used in place of O2, because it can
act as an O2 mimic and, upon binding, produces a stable. EPR-active complex. By replacing the
hydrogens normally targeted by the ferryl complex with deuteria, the hyperfine coupling between
the Fe(III)-NO iron complex and the deuterium could be used to infer geometric information. On
the basis of this analysis, the angle between the Fe-N bond and the Fe-2
H vector was determined
to be nearly 90°. [5] It is this positioning that may be primarily responsible for the very slow H•-
abstraction rate in the chlorination reaction. [26]
19. 12
The hypothesis of a perpendicular alignment of the Thr methyl C-H bond and the O2–
of
the ferryl complex in the SyrB1•SyrB2 complex provides important information about how the
active site of SyrB2 might look during the chlorination reaction. Prior to this research, many
configurations of the initial Fe(III)-superoxide adduct were considered, and the three shown in
Figure 1-5 (illustrated with NO in place of O2) were believed to represent the most likely possible
points at which O2 could add to the iron(II) cofactor. However, only two of these arrangements
[Figure 1-5 (B) and (C)] would obviously take into account the positioning hypothesis, making
the left structure (A) unlikely, as will be described further in the next section.
Figure 1-5. Possible Geometries of O2 Addition During the Chlorination Reaction of SyrB2.
The figure (From Martinie et al. JACS 2015 [5]) shows the outcome of replacing water (A),
chloride (green) (B), or the 2OG carboxylate (C) with NO (blue and red 2-atom molecule), the
mimic of O2 that produces the EPR-active iron nitrosyl species. (A) is inconsistent with the N-Fe-
2
H angles determined experimentally in that study. In (B) and (C), the NO group is shown
approximately perpendicular to the Thr methyl group. (B) and (C) show the two possible
locations for O2 during the chlorination reaction. [5]
20. 13
Figure 1-5 (B) is the first of two theoretically possible configurations of the SyrB2 active
site. In this model, O2 (in the actual reaction, as opposed to NO in this experiment) would
effectively displace the chloride in the initial O2 adduct of the chlorination mechanism. The
depicted shift in the location of the chloride ligand to the top, axial location would make it
possible for the SyrB1 arm to extend into the active site either from the side of the chloride ligand
of from the side of the 2OG carboxylate. Figure 1-5 (C), showing the replacement of C1 of 2OG
with NO, models the other possible configuration of the active site during the chlorination
reaction. In this model, C1 of 2OG shifts into the top axial location so that O2 takes up the
position vacated by the carboxylate. This model could create significant steric challenges, as the
bulky carboxylate could clash with the SyrB1 phosphopantetheine arm if located at the top axial
site. [5]
To this point, no experimental data has been available to assess how the SyrB1
phosphopantetheine arm enters the active site of SyrB2 to present the amino acid for chlorination.
Referring to Figure 1-5 (B), the entry point could be either from the side of the chloride ligand or
from the side of the 2OG carboxylate. Evidence pointing to entry from the 2OG-carboxylate side
molecule would likely rule out the configuration of Figure 1-5 (C) as a viable possibility, as the
interaction of 2OG and the substrate would likely prohibit chlorination. [5] This thesis describes
initial work toward clarifying the trajectory of the phosphopantetheine arm of SyrB1 as it presents
the target amino acid to SyrB2 for chlorination. The results are expected to explain how the
enzyme achieves the required substrate positioning that prior work has implicated as the key to
directing selective chlorination and avoiding hydroxylation.
21. 14
Chapter 2
Materials and Methods
Chapter 2.1 Materials
Primers were ordered from Integrated DNA Technologies for SyrB2 and SyrB1 DNA
plasmid Polymerase Chain Reaction (PCR) modification. Primers ordered are listed
below (Table 2-1 and Table 2-2).
Primers Ordered and Used for SyrB2:
Change in Amino acid
sequence
Forward DNA Sequence Reverse DNA Sequence
Cys157 Ala F- 5’- GCC CTG CAG
TTC ATT CCC GGC -3’
R- 5’- ACC ATT GGC
AAT GTT CGC GTC -3’
Cys98 Val F- 5’- GTG TGG CGT
ACC GAG TTC TTT CCC
-3’
R- 5’- GAG CAC GTT
CGG GCC G -3’
Cys85 Ser F- 5’- GAA ATC TCC
GAT CGC GTC GAA
AGC -3’
Cys80 Ser R- 5’- CGG GCG AGA
GAT GTG GCT GG -3’
22. 15
Table 2-1. DNA Primer Sequences Used in "Around-the Horn" Mutagenesis of SyrB2
The table above shows the custom DNA primer sequences used to remove native cysteines and
add new cysteine residues. The codons for all native cysteines were changed to encode Ser or
Ala, and codons for four new Cys residues were introduced. Each variant had exactly one new
cysteine residue added at either position 18, 47, 245, or 258. *Indicates the sets of primers used to
Gln245 Cys F- 5’- TGC GAA ATG
CGC ATG GGC TTC G -
3’
R- 5’- TGA TTC GCC
ACT GTG CGG -3’
Ser47 Cys F- 5’- TGC GCT GCC
GCC TAC C -3’
R- 5’- ACG GTC GAG
CAG GCG C -3’
Ser258 Cys F- 5’- TGC TTC GTC
CAT GTC TAC CCG
GAT TC -3’
R- 5’- GGG TAC ATA
GCG TGA CGC G -3’
Asn18 Cys F-5’- TGC GGT TTT ATC
GGG CCG TTT G -3’
R-5’- TTT CTC GAA
CGA GGC ACG C -3’
Thr143 Ala* F-5’- GCT GTC TGG
ACC GCA TTT ACC G -
3’
R-5’- AAT GGT GCC
GCC GAA CTC -3’
Ser231 Ala* F-5’- GCG ACG CTG
ATG CAC GC -3’
R-5’- CCA GAA AAT
AAT GAA CTG CCC GG
-3’
23. 16
create SyrB2 variants outside the scope of this project. However, these primers are listed because
these variants were made at the same time and with the same procedure as the variants prepared
for this project.
Primers ordered and used for SyrB1:
Table 2-2. DNA Primer Sequences Used in "Around-the-Horn" Mutagenesis for SyrB1
The above table shows the primers that were used to change the codons in the gene for the SyrB1
protein. Two codons were modified. The first modification replaces a native Cys residue with a
non-spin-active residue. After the first modification is confirmed, a second modification
introduces a new, non-native Cys residue onto the already-mutated plasmid.
Chapter 2.2. Polymerase Chain Reaction (PCR) Mutagenesis
Chapter 2.2.a. PCR Reaction Mix
A sample of the plasmid encoding the His6-tagged SyrB2 protein and conferring
kanamycin resistance was obtained from Penn State Research Associate Dr. Benjamin Allen. A
Change in Amino acid
sequence
Forward DNA Sequence Reverse DNA Sequence
Cys419 Ser F-5’-TCC GAC GAG
CAA TTG AAA ATC
AGC -3’
R-5’-GCG ACC GGC
ATA ACG GTA TTC -3’
Thr582 Cys F-5’- TGC CAT TAC
AAA ATCAAC CTG
GAC CCC -3’
R-5’- CTT GAG CTT
GCT CAG CGA GC -3’
24. 17
sample of the plasmid encoding the His6-tagged SyrB1 protein and conferring kanamycin
resistance was obtained from Penn State Postdoctoral Scholar Dr. Wei-chen Chang. Initially, a
50 µL mix was created based on Dr. Allen’s previous SyrB2 mutagenesis. The recipe for this mix
is given in Table 2-3. The aforementioned SyrB1- and SyrB2-encoding plasmids were used as
templates in the PCR reactions.
Chapter 2.2.b. PCR Protocol
PCR Mix Ingredient Amount
Required (µL)
Sterile water 33
5x Q5, taq buffer 10
10 µM forward primer 2.5
10 µM reverse primer 2.5
dNTPs 1
Template plasmid, 0.5
ng/µL
0.5
Q5 polymerase 0.5
Total 50
Table 2-3. Mix for “Around-the-horn” site-directed mutagenesis via PCR
25. 18
The above table shows the initial PCR mix recipe that failed to remove the cysteine codon from
the desired position in the plasmid. Water had been previously autoclaved under sterile
conditions. Buffers used correlated with the chosen polymerase for PCR. For example, Q5
polymerase was used with a 5x Q5 buffer. Forward and reverse primers were both used in the mix
to ensure that the cysteine was changed at the desired location in both the 5’ to 3’ and 3’ to 5’
strands of the plasmid. Primers used in experiments are given in Chapter 2.1. dNTPs, or
deoxynucleotide triphosphates, are DNA building blocks that a polymerase can add to the
growing DNA strand. Q5 and taq were chosen as the initial polymerases to use in the reaction.
However, the experimental procedure required an optimization of this process, and so the initial
procedure was modified from the original source to produce the desired PCR product. (See
Chapter 2.2.c.)
Step Number Temperature for Step Time
1 98° C 30 s
2 98° C 10 s
3 62° C 20 s
4 72° C 3 min
5 Repeat Steps 2-4 for
35 Cycles
3 min 40 s x 35
cycles
6 72° C 2 min
7 4° C Forever- for storage
Table 2-4. PCR steps for Bio-Rad C1000 Thermal Cycler
26. 19
The PCR steps used for mutagenesis are shown above. Initial annealing temperature was 72° C
with an extension time of 3 min. Modifications to this procedure were made in accordance with
the polymerase used. See Chapter 2.2.c.
Chapter 2.2.c. PCR Modifications
Following several unsuccessful trials with different polymerases and annealing temperatures, a
200 µL PCR mix recipe was created and proved to be the solution to earlier problems with the
polymerase chain reaction.
PCR Mix Ingredient Amount Required
(µL)
Sterile water 133
5x Phusion buffer 40
10 µM forward primer 10
10 µM reverse primer 10
dNTPs 4
Template plasmid, 0.5 ng/µL 2
Phusion Polymerase 1
Total 200
Table 2-5. Modified PCR Mix
27. 20
The above table gives the PCR mix that was used to successfully create all SyrB1 and SyrB2
variants. The 200 µL mix was used to create four 50 µL samples that would have different
annealing temperatures. This mix also used Phusion polymerase. Template plasmid concentration
was often used at concentrations greater than 0.5 ng/µL. Most often, the template plasmid was
prepared by 1:10 or a 1:100 dilution of the stock solution with sterile water.
Additionally, to find an effective annealing temperature, a temperature gradient was
programed into the thermal cycler. The gradient was set so that the minimum temperature was
64° C and the maximum was 72° C. 50 µL aliquots of PCR mix were staggered (Table 2.6).
72
71
70
69
68
67
66
65
64
Table 2-6. Annealing Temperature Gradient with Markers for Samples
28. 21
The table shows the gradient pattern used for the PCR annealing step. Samples were annealed at
the temperature boxes that contain circles (temperatures 65, 67, 69, and 71° C). All other steps of
the reaction followed the protocol described in Chapter 2.2.a.
Chapter 2.3. Agarose Gel Electrophoresis
A 1.5% agarose gel was prepared using 1 mL of 50x Tris/acetate/EDTA (TAE) Buffer for
every 50 mL of water. 100 mL of 1X TAE solution was prepared at a time in a 250 mL
Erlenmeyer flask. The solution was autoclaved on the liquid cycle of a Beta Star Corporation
Model 2CO2BS Autoclave. When the gel was approximately 30-40° C, 5 µL of Ethidium
bromide was added to 50 mL of solution. Because not all of the agarose solution was used at one
time, the remaining agarose solution, which solidifies at room temperature, was heated in a
microwave oven until it became completely liquid. At 30-s intervals, heating was terminated, and
the flask was swirled to distribute heat. This process was continued for 1-2 min of total heating
time or until the agarose solution started to boil. After the solution had cooled down to 30-40° C,
ethidium bromide was then added to the solution. The agarose solution was stirred by gently
swirling the flask. The solution was slowly poured into an agarose gel tray and a comb was placed
into the gel to create wells for the DNA. After 20-30 min at room temperature, the gel solidified
and turned a translucent white color. The gel was removed from the tray holder and placed onto
the electrophoresis unit. PCR product DNA samples were mixed with agarose loading buffer in a
ratio of 10 µL of sample to 2 µL of loading buffer in an Eppendorf tube. A 5-10 µL aliquot of a
DNA molecular weight ladder mix was pipetted into the first well of the agarose gel. The DNA
sample and loading buffer solutions were mixed with a pipet until homogenous. The samples
were then pipetted into the subsequently open wells on the gel until each well was full. The
29. 22
electrophoresis unit was filled with 1X TAE Buffer and closed with the unit’s lid. The lid was
aligned so that the red terminal connected with a red electrode and the black terminal connected
to the black electrode. The red and black wires were connected to the corresponding black and red
ports on the voltage source. The voltage was set to 120 mV, the current was set to 500 amps, and
time was set to 60 min on the power source. During the electrophoresis run, DNA samples stained
with the agarose loading buffer moved down the length of the gel toward the positive red
electrode. Once the time was completed or the loading dye showed that the DNA had moved the
entire way across the gel, the gel run was stopped. The gel was removed from the electrophoresis
unit and observed under UV light. Bands visible in the rows associated with each DNA well
indicated a successful PCR amplification of double-stranded DNA.
Chapter 2.4. Phosphorylation/Ligation (KL) Reaction
After PCR was completed and gel analysis showed probable amplification, the PCR product was
ligated with T4 DNA Ligase (Table 2-7). The total reaction required a volume of 10 µL. After all
ingredients were added, the reaction was pipetted slowly up and down several times to mix. The
reaction was then allowed to incubate at room temperature for a minimum of twenty min.
KL Reaction Ingredient Quantity in Reaction (µL)
Sterile Water 7
100x-diluted PCR reaction 1
10x T4 DNA Ligase reaction buffer 1
T4 PNK 0.5
T4 Ligase 0.5
Total Reaction Mix 10
Table 2-7. KL Reaction Mix
30. 23
The table shows the ingredients of the reaction to ligate the PCR product with T4 DNA Ligase.
Ligation was required so that the disjoined PCR products could be linked by phosphodiester
bonds into circular DNA plasmids to allow transcription to occur successfully within E. coli.
Chapter 2.5. Transformation of the Ligated PCR Product
KL Reaction Product was used to transform antibiotic-sensitive to antibiotic- resistant
competent XL-1 Blue/ DH5 Alpha E. coli cells. These cells are known as "competent" because
they have been treated to permit antibiotic-resistance encoding DNA to be taken up and
replicated. To transform, a pre-made aliquot XL-1 Blue E. coli cells was removed from a -80° C
freezer and immediately placed on ice. While the cells thawed, a Lauda Alpha A6 water bath was
heated to 42° C. After a minimum of three min, the cells melted to have a slushy consistency that
could be easily stirred with a pipette. Once this consistency was reached, 3-5 μL of KL Reaction
product was added to the XL-1 Blue competent cells under sterile conditions. Sterile conditions
require cleaning the lab surface with ethanol, lighting a Bunsen burner, and autoclaving pipette
tips on a solid cycle. DNA was exposed to air, and thus to potential contaminants, for as little
time as possible.
After adding the DNA, the cells were pipetted up and down with a sterile tip and left on
ice for 2 min. After this time, the cells were placed in the 42° C water bath for forty-five seconds.
The cells were then again placed on ice for three to five min. A 1 mL aliquot of sterile LB
medium (5 g tryptone, 2.5 g yeast extract, 2.5 g NaCl in 250 mL of solution; adjusted pH to 7.3±
0.1 with sodium hydroxide; autoclaved on media cycle) was then added under sterile technique to
the tube with the cells followed by brief mixing with a pipette. The cells were placed into a 37° C
incubator for 45-60 min. After the allotted time, the cells were pelleted by centrifugation for 30 s
at 11,000 g. The supernatant was removed under sterile conditions until about 100 µL remained.
The cell pellet was then resuspended with a sterile pipette tip in the remaining supernatant. Once
mixed, the solution was transferred by pipette onto a LB-kanamycin plate. (2.5 g tryptone, 1.25 g
31. 24
yeast extract, 1.25 g NaCl; adjusted pH to 7.3± 0.1 with Sodium Hydroxide; added 3.5 g of
molecular biology grade agar; adjusted volume to 250 mL; autoclaved on media cycle; solution
allowed to cool; added 250 µL of Kanamycin solution [50 mg/ mL solution made from a stock
powder and then refrigerated]; poured into unopened petri dishes under sterile conditions; dishes
exposed to Bunsen burner flame to remove air bubbles and then were sealed and refrigerated.)
Using a glass spreader (either an L-shaped glass rod or a glass pipette that was melted into an L-
shape using the Bunsen burner) that was sterilized with the flame of the Bunsen burner, the cell
solution was spread across the whole surface of the plate. The plate was then placed into the 37°
C incubator overnight. In the morning, the plate was removed, sealed with Parafilm, and placed in
a 4° C refrigerator. In the afternoon, a single E. coli colony was removed from the plate by using
a sterile tip under sterile conditions. The tip with the colony was ejected into a sterile Falcon tube
containing 10 mL of sterile LB media and 10 μL of 50 mg/ mL Kanamycin. The lid was
unscrewed slightly and then fastened with a tape seal so that air could enter the tube while it was
shaken but the lid would not fall off the container. The tube was allowed to shake at 180 rpm at
37° C overnight.
Chapter 2.6. DNA Extraction
Chapter 2.6.a. Initial DNA Extraction and Concentration
The next morning, following growth of the culture, the cells were removed from the
shaker for DNA extraction. DNA extraction followed a standard (Macherey-Nagel Nucleospin
Plasmid- 5.2- Isolation of Low-Copy Plasmids) [27]. Through practice, several changes were
made to this procedure that increased DNA yield significantly and are discussed in section 2.6.b.
DNA was obtained according to this procedure, and the expected concentration was at least 150
nanograms of DNA per µL of solution. The DNA was measured using a Thermo Scientific
32. 25
NanoDrop 2000c UV/VIS Spectrophotometer. To use the spectrophotometer, the program was
opened on a laptop. The DNA sample analysis tab was selected. 1 µL of Nanopure distilled water
was used to clean the analysis podium and another 1 µL was used as a blank. The lid was closed
over the podium and “blank” was selected on the program to run. Then, 1 µL of DNA solution
was placed on the podium and “sample” mode was selected on the program to run. The podium
was cleaned after the measurement, and an additional 1 µL of DNA solution was used as a second
sample. If the concentrations indicated by the two replicate measurements were similar, the
average of the two values was taken to be an adequate estimate of the DNA concentration. If the
values diverged by more than 20%, additional measurements were taken and included in the
average. If the DNA concentration was found to be less than 100 ng/µL, the DNA solution was
concentrated further by using a SpeedVac. To run the SpeedVac, an Eppendorf tube containing
the DNA solution was placed into the machine with the lid open. The lid was then closed and the
spin mode was switched on. When the temperature in the chamber reached approximately -80° C,
the vacuum was switched on. The SpeedVac was allowed to run for approximately ten min, and
then the DNA concentration was then re-determined using the NanoDrop spectrophotometer. The
solution was reconcentrated until the DNA reached a minimum of 100 ng/µL.
Chapter 2.6.b. Modifications to DNA Extraction Protocol
Step Original Procedure Modification
Add A1 Buffer Use 500 µL Use 750 µL
Add A2 Buffer Use 500 µL Use 750 µL
33. 26
Tube Inversion with
A2
Invert 6-8 times Invert until sample is clear and uniform
Add A3 Buffer Use 600 µL Use 900 µL
Tube Inversion with
A3
Invert 6-8 times Invert until color changes completely from
blue to clear
Wash Silica
Membrane
Add optional buffer
AW
Always add buffer AW
Drying Time 2 min 8-10 min
Elution Buffer (AE) Preheated to 70° C Kept at room temperature
Elution Single Elution Double Elution- repeat elution step
Table 2-8. Modifications to DNA Elution Procedure
The table above depicts changes made to the Macherey-Nagel Nucleospin Plasmid- 5.2- Isolation
of Low-Copy Plasmids protocol. Effects of these changes are discussed in depth in Chapter 4.1.
Chapter 2.7 Sequencing
When an appropriate concentration was reached, 5 mL of each unique sample was
placed into two new Eppendorf tubes. This sample was then used in forward and reverse
sequencing introduction of the desired substitution without any additional unwanted
modifications to the gene sequence of SyrB1 or SyrB2. A sequencing request form was submitted
34. 27
online and a receipt of the request was printed for lab records (Figure 2-1). The samples were
given to the Penn State University Nucleic Acid Facility Sanger DNA sequencing lab for
sequencing (406 Chandlee Lab; University Park, PA; 16802).
Figure 2-1. DNA Sequencing Request Form
The above form is a DNA Sequencing request form that was used for tracking DNA samples that
were submitted for sequencing.
The sequencing results were processed within twenty-four hours and were available for
viewing and downloading online. The sequencing results appeared as a list of DNA nucleobases
and were translated to read the corresponding amino acid sequence using a translate tool. Once
35. 28
translated, the results were compared with the appropriate wild type SyrB2 amino acid sequence,
Q9RBY6-1 [28]. The SyrB1 sequence used was Q52400-1 [29]. The amino acid sequences had to
be identical to continue on with the experiment. Any discrepancies in the sequence indicated that
unwanted substitutions had been introduced, and any DNA samples that contained additional
substitutions were discarded. When unwanted substitutions occurred, the mutagenesis process
was repeated (optimized steps described in Chapter 2.2.a. through Chapter 2.7 were repeated).
Error-free samples were then used to continue the cysteine-removal process.
Chapter 2.8. Preparation of New BL21 Competent Cells
New competent BL21 DE3 E. coli cells were prepared for expression of the S258C
variant protein. Preparation of competent cells followed a standard lab procedure that is included
as Appendix A. All buffers were prepared, stored, and used at 4° C. Once the buffers were
prepared, the cells were tested for contamination from surrounding plasmids. To test for
contamination, two frozen aliquots of cells were removed from the -80° C freezer and placed on
ice. Each was transformed as previously described; however, a DNA plasmid conferring
kanamycin resistance was added to only one of the two samples. The sample with the plasmid
served as a positive control to test transformation competency, and the sample without the new
plasmid served as a negative control to ensure against contamination. The samples were plated
onto two kanamycin plates and stored in a 37° C incubator overnight. The cell stock was
determined to be competent if cells grew on the plate containing the positive control but no cells
grew on the plate containing the negative control sample.
36. 29
Chapter 2.9. Protein Growth
DNA samples encoding Cys variants of SyrB2 were used to transform the competent BL21 DE3
cells. Plasmid was added, and the cells were incubated for one hour at 37° C. The cells were then
pelleted by contrifugation for thirty seconds at 11,000 g, re-suspended in 150 µL of LB medium,
and spread onto an LB agar kanamycin plate. The plate was left in the incubator overnight and
then stored at 4° C until afternoon (minimum of 12 hours of incubation time). A 250 mL starter
culture was prepared by autoclaving 250 mL of LB media, cooling, and adding 250 µL of 50 mg/
mL kanamycin. A single E. coli colony was scraped off the LB agar kanamycin plate and used to
inoculate the starter culture. The starter culture was placed in a New Brunswick shaker at 200 rpm
and 37° C overnight. The next morning, the culture was removed. For the Q245C variant-
containing cells, twelve 2.8 L flasks containing 1 L of LB medium in each were prepared and
autoclaved using the media cycle. A 12 mL aliquot of starter culture was added to each flask
under sterile conditions. For the S258C variants, six 1 L portions of LB medium were prepared in
2.8 L flasks. The flasks were again autoclaved in the media cycle and then cooled. A 20 mL
aliquot of the starter culture of the SyrB2 S258C variant strain was added under sterile conditions
to each of the six media flasks. The flasks were allowed to shake at 200 rpm in the New
Brunswick and Fischer Scientific MaxQ 5000 incubators at 37° C until the cultures reached an
optical density at 600 nm (OD600) of 0.6-0.8. Culture OD600 was determined by using a WPA
Biowave CO 8000 cell density meter. After referencing with water, a 1-2 mL sample of culture
was withdrawn for measurement. At the target OD600, cultures were induced to express the
desired protein by addition of 0.12 g isopropyl β-D-1thiogalactopyranoside (IPTG). Cultures
were shaken at 200 rpm and 18° C overnight. The next morning, the culture was centrifuged in a
Beckman-Coulter at 8,000 g for 20 min. The supernatant was removed, and the cells were frozen
in pellets in liquid N2. The cell pellets were stored at -20° C.
37. 30
Chapter 2.10. Purification
Chapter 2.10.a. Initial Purification Procedure
Frozen cells pellets were weighed and resuspended in approximately five times their
mass of lysis/purification buffer (500 mM NaCl, 50 mM 2-[4-(2hydroxyethyl)piperazin-1-
yl]ethanesulfonic acid (HEPES), 10mM imidazole; pH 7.5). The cells were stirred at room
temperature for five min to completely resuspend them. A 200 µL aliquot of of 1 M
phenylmethane sulfonyl fluoride (PMSF) stored in anhydrous isopropanol (1µL PMSF: 1 mL
cell-containing media) to prevent protein sample degradation, and the mix was then stirred at 4° C
at a more vigorous rate. A 0.20 solid sample of lysozyme and 0.02 g of deoxyribonuclease
(DNAse) (1 mg lysozyme, 0.1 mg DNAse per 1 mL of cell suspension) were added and could be
seen to become homogeneous after approximately five min of stirring at 4° C. The stirring
continued for twenty min afterwards. A column packed with Ni(II)-nitrilotriacetate (NTA)
agarose resin was allowed to come to equilibrium with the same lysis/purification buffer during
this time, and a Fischer Scientific 550 Sonic Dismembrator (sonicator) was set up. The sonicator
was programmed to run for six min and forty-five seconds of pulse time. The sonicator was set to
pulse for forty-five seconds, then wait two min, then repeat for the duration of the program. The
cell suspension was placed in a metal cup within an outer dish containing ice and stirred. The
sonicator was situated with the sound emitting tip inside the inner metal cup. The sonication
program ran for the allotted time, and a centrifuge was allowed to cool to 4° C. The sonicated
solution was then poured into a centrifuge tube, which was balanced with a matched tube filled
with water. The containers were placed in a JA 14 centrifuge rotor and spun at 22,000 g on the J-
26 XP for twenty min. The supernatant was then poured carefully onto the nickel column. The
solution was allowed to drip slowly out of the column while the eluate was collected in a beaker.
The column was washed with five volumes of this same lysis/purification buffer. After washing,
38. 31
approximately four column volumes of elution buffer (50 mM HEPES, 200 mM Imidazole; pH
7.8) were added to the column. As the elution buffer passed through the column, the eluate was
collected and poured into several empty Amicon Ultra or Macrosep Advance 10 kDa Molecular
Weight centrifuge concentrator tubes. The tubes were centrifuged in a Thermo Scientific/ Sorvall
Legend RT tabletop centrifuge at 3500 g. When the retained volume was less than ~ 20 mL total,
it was consolidated for dialysis.
Chapter 2.10.b. Modified Protein Expression and Purification through Expression Tests
Modifications were made to the standard expression and purification procedures toward
the goal of optimizing yield of SyrB2 obtained in the purification. Variables used in the
expression tests included incubation time at 37° C and the OD600 at the time of induction of the
cultures of the SyrB2 S258C variant expressing strain, concentration of IPTG used to induce
expression, time of incubation after induction, and lysis/purification and elution buffers used.
Variables and controls are listed below (Table 2-9).
Variables in Trial Absorbance
Reading
Before
Induction
(OD600)
Amount of
IPTG
[Concentration]
Time of
Sample
After
Induction
Buffers used
Control 0.6-0.8 120 mg
[0.5 mM]
20+ hours Standard
equilibration and
elution; see
Chapter 2.2.i.
39. 32
Varied Absorbance,
IPTG, and Time After
Induction
0.3-0.4 or 0.6-
0.8
6, 30, or
120 mg
[0.025, 0.125,
and 0.5 mM]
4 and 20+
hours
Cells not purified
Varied Absorbance,
IPTG, and Time After
Induction; new buffers
0.4-0.5 6, 30, or
120 mg
[0.025, 0.125,
and 0.5 mM]
3.5, 5, and
20+ hours
Equilibration
and elution
buffers with
glycerol and KCl
Varied IPTG, and Time
After Induction;
samples removed from
shaker after final three
time points; new
absorbance and buffers
0.4-0.5 6 or 20 mg
[0.025 or 0.083
mM]
4, 6, 7.5, 21
hours
Equilibration
and elution
buffers with
glycerol and KCl
Table 2-9. Variables and Control Values in Expression Test Trials
As described in Chapters 2.10.a., the purifications from BL21 cells typically involved
induction at OD600 ~ 0.6-0.8 by addition of IPTG to a concentration of 503 µM, and cell lysis and
protein purification with pH-adjusted HEPES buffers. I attempted induction at less values of
OD600 in the hope that more protein would be produced or a greater fraction of the protein
produced would be soluble. Lesser concentrations of IPTG in the induction were also attempted
to determine the minimum concentration needed for induction. The reason for this attempt is that
it is often true that slower production of the desired protein results in a greater fraction that is
properly folded. After induction, samples of culture were collected at various times to determine
the kinetics of protein expression. Buffer content was changed to attempt to stabilize the SyrB2
variant proteins. Equilibration and elution buffer changes will be described later in this section. In
the second expression test, cells were rapidly chilled immediately after addition IPTG by
40. 33
immersing the culture flasks in ice. In the second and third expression tests, OD600 at induction
was 0.4-0.5, and, because these trials gave good results, this became the target OD600 at which to
induce. .
In each expression experiment, samples were collected for determination of protein
production by analysis on SDS PAGE gels. An aliquot was removed from each culture flask;
aliquot volumes were scaled so that the same quantity of cells would be used in each case. The
culture samples were then pelleted in a microcentrifuge for 1 min at 11,000 g and placed in the
freezer. Before SDS PAGE analysis of the aliquots, the cell pellet was resuspended by pipetting.
The sample was then rapidly frozen and thawed by submersion in liquid nitrogen followed by
submersion in 50° C water. This freeze-thaw step was repeated five times to ensure complete lysis
of the culture sample. A 10 µL aliquot of the resuspended pellet was mixed with 10 µL of SDS
PAGE loading dye (pre-made by Thermo-Scientific; contains 40% glycerol, 4% lithium dodecyl
sulfate, 4% Ficoll 400, 0.8 M triethanolamine-Cl [pH 7.6], 0.025% phenol red, 0.025% coomassie
G250, 2 mM EDTA disodium) and the mixture was incubated in boiling water for 4 min. The
samples were then suitable to be loaded on the gel.
In the first round of expression tests, OD600 at induction and concentration of IPTG were
varied. Six 1 L cultures with LB medium were inoculated with approximately 20 mL from a
started culture of the E. coli strain expressing the SyrB2 S258C variants, as described in Chapter
2.9. Each flask was either induced at the control OD600 of 0.6-0.8 or at lesser value of 0.3-0.4.
IPTG was added to each flask to a final concentration of either 25 µM, 125 µM, or 503 µM. The
contents of each flask were organized in the New Brunswick Excella E25 incubator to allow
multiple variables to be tested at once (Table 2-10).
41. 34
Absorbance at induction: 0.6-
0.8
IPTG: 6 mg [25 µM]
Absorbance at induction: 0.6-
0.8
IPTG: 30 mg [125 µM]
Absorbance at induction: 0.6-
0.8
IPTG: 120 mg [503 µM]
Absorbance at induction: 0.3-
0.4
IPTG: 6 mg [25 µM]
Absorbance at induction: 0.3-
0.4
IPTG: 30 mg [125 µM]
Absorbance at induction: 0.3-
0.4
IPTG: 120 mg [503 µM]
Table 2-10. Absorbance and IPTG Amount for First Expression Test
The table shows the OD600 values and the concentration of IPTG used in each flask during the
first expression test. Each rectangle corresponds to a flask containing 1 L of culture. The controls
had OD600 = 0.6-0.8 and 120 mg [503 µM] IPTG in the inductions. Samples were taken from each
flask 20+ hours after induction. The gel results are shown in Chapter 3.3.
A second round of expression tests was completed. In this round, multiple parameters
were varied: i.) amount of IPTG used (6, 30, or 120 mg [25, 125, or 503 µM]) ii.) cells were
chilled prior to induction iii.) the number of time points post-induction was increased. During
purification, different buffers were used for equilibration and elution (Table 2-11). Purification
was modified for a smaller quantity of cell mass, and a smaller Ni(II)-NTA agarose column was
used for elution. Additionally, cells were lysed not by sonication but by three passages through a
French pressure cell at 292.3-314.7 PSI.
42. 35
Equilibration buffer
50 mM HEPES
20% vol/vol glycerol
300 mM KCL
10 mM Imidazole
Adjust pH to 7.5 at 4° C
Elution Buffer
50 mM HEPES
20% vol/vol glycerol
300 mM KCL
200 mM Imidazole
Adjust pH to 7.5 at 4° C
Table 2-11. Modified Equilibration and Elution Buffers
20% glycerol (by volume) and 300 mM KCl were added to the recipes for the equilibration and
elution buffers. These ingredients were used in the hope of stabilizing the variant proteins.
In the third expression test, six 1 L cultures were again grown. However, in this test,
flasks were removed entirely from the shaker after each of the final three time points; samples
were not simply taken for gel analysis. The OD600 at the time of induction was 0.4-0.5. Either 6 or
43. 36
20 mg of IPTG was used in each flask [25 or 83 µM] . IPTG concentration and time of incubation
after induction were again varied (Table 2-12).
Table 2-12. IPTG Concentrations and Time of Incubation after Induction in the Third Expression
Test
This table shows the parameters used in the third expression test. Flasks were removed from the
shaker at the indicated time (following induction by addition of IPTG).
Chapter 2.11. SDS-PAGE Electrophoresis
A sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) apparatus
was used to determine if protein was expressed and if protein was successfully purified. The
recipe for the gel is listed below (Table 2-13). Once the gel was solidified, it was removed from
the outer holder while still fastened between two glass plates. The gel was then clamped upright
into an SDS-PAGE gel apparatus with the wells opening upward. SDS-PAGE buffer (30 g Tris,
144 g glycine, 10 g sodium dodecyl sulfate in 1 L; pH 8.3; amount used was diluted 1:10 by
volume prior to use) was poured onto the apparatus, filling the tray and the opening behind the gel
to the top. The apparatus was then attached to a voltage source with positive and negative
IPTG: 6 mg IPTG: 6 mg IPTG: 6 mg
End time point: 6:30 PM End time point: 8 PM End time point: 9 AM
IPTG: 20 mg [83 µM]
End time point: 6:30 PM
IPTG: 20 mg [83 µM]
End time point: 8 PM
IPTG: 20 mg [83 µM]
End time point: 9 AM
44. 37
terminals connecting to corresponding to ports on either side of the gel. A Bio-Rad Precision-Plus
Protein Dual Color Standard ladder was loaded into the first lane, then the remaining lanes were
filled with samples combined with SDS-PAGE loading dye (previously described). The voltage
was set to 170 mV, and the gel was allowed to run one hour. The gel was removed from the
apparatus and glass plate holders. The gel was then placed in a Tupperware container. Next, the
gel was briefly washed with DI water. Coomassie brilliant blue staining buffer (pre-made,
recycled) was then added to the container. The gel was next heater in a microwave oven for thirty
seconds or until the staining solution began to boil. The gel was then removed from the
microwave and shaken for ten min. The staining buffer was poured into a storage container for
reuse. Next, the stained gel was rinsed with DI water. After this rinse, destaining buffer (40%
ethanol, 10% glacial acetic acid) was poured into the Tupperware container. The container was
then heated again in the microwave oven for ~ 30 s and then shaken on the shaking plate for 10
min. Finally, the destain solution was discarded and the gel was stored in DI water. Bands in each
layer became distinguishable after several hours of immersion in water.
Clear Gel Phase
Ingredient Amount
24% Acrylamide Solution 4 mL
0.78 M Tris pH 8.8 Buffer
TEMED and SDS pre-added
4 mL
Gelling solution 1 capful
45. 38
Blue Gel Phase
0.25 M Tris-HCl pH 6.5
510 µL SDS, 500 µL TEMED pre-
added
2 mL
10.2% Acrylamide Solution 2 mL
Gelling Solution 1 capful
Table 2-13. SDS-PAGE Acrylamide Gel Recipe
The table gives the ingredients necessary for each phase of the acrylamide gel.
The clear phase ingredients of the gel were mixed in a 15 mL falcon tube. Then, the
mixture was pipetted onto an acrylamide gel caster. A layer of ethanol was poured on top of the
gel. The gel was allowed to polymerize for 45 min. At this point, the ingredients of the blue layer
were mixed in a 15 mL falcon tube. The ethanol layer was poured out from the gel caster. Then,
the blue solution was pipetted onto the caster until no space remained within the gel compartment.
A plastic comb was then placed vertically into the blue layer to form wells. The gel was allowed
to solidify for at least 30 min before it was used.
Chapter 2.12. Dialysis
A strip of 5.9 or 8.1 mL/cm dialysis tubing was cut and soaked in room-temperature
water. Water was then poured through the tubing to open it for use. One end of the tubing was
clamped tightly and the protein solution was pipetted into the dialysis tube. The protein solution
was poured into the dialysis bag. The open end of the tubing was clamped so that an air bubble
46. 39
filled some of the open space in the bag. The bag was placed in a 4 L beaker containing an
imidazole-free equilibration buffer (50mM HEPES, 1mM EDTA buffer, [pH 7.5] 4°C) overnight
and allowed to stir gently with a magnetic stir bar in a 4° C refrigerator room (cold room). The
next morning, the dialysis bag was moved into a different 4 L beaker containing fresh 50 mM
HEPES Buffer and gently stirred in the cold room. After two or three hours, the dialysis bag was
again changed to fresh buffer. The bag was again allowed to dialyze for at least 2 h while stirring
gently in the cold room.
Chapter 2.13. UV/VIS Spectroscopy and Protein Concentration
A minimum of four Eppendorf tubes were prepared with protein samples for analysis by
UV-visible absorption spectroscopy. A 400 µL aliquot of 50mM HEPES (pH 7.5) buffer was
added to each tube. The first tube served as a blank. 1 or 4 µL of protein solution was added to
each of the other three tubes to make 1:400 or 1:100 diluted samples for the analysis. If the
protein solution was highly concentrated with protein, dilutions of 1:400 were required to stay
within the linear range of the spectrophotometer. An Agilent Technologies Cary 8454 UV/VIS
Spectrometer was turned on and used for analysis. After powering on and opening the software on
the desktop, the spectrometer was allowed to warm up for a minimum of twenty mins. When the
spectrometer was ready, a green light appeared on the front of the apparatus. On the left side of
the program window, the parameters for the spectroscopic reading were set (Table 2-14).
Type of Reading: Single Reference Wavelength
47. 40
Wavelength: 280 nanometers (nm)
Background Correction: 800 nm
Spectrum Range: 250-800 nm
Table 2-14. UV/VIS Spectroscopy Settings
The table shows the settings for UV-visible absorption measurements. 280 nm was chosen as the
selected wavelength because the amino acids tyrosine and tryptophan, both present in the SyrB1
and SyrB2 amino acid sequences, absorb in this spectra region.
A glass pipette was used to transfer the “blank” HEPES solution into a glass cuvette designed for
use in the spectrometer. A clamp built into the spectrometer was used to fasten the cuvette in
place. The blank button on the program window was selected to set the protein-free sample as a
standard. When a spectrum for the blank appeared in a new window, the cuvette was removed
from the instrument and emptied.
The cuvette was cleaned before the next measurement. A vacuum cuvette washer and
dryer was used for cleaning. To use the cleaning device, the opening on the beaker was connected
to a vacuum faucet. The cuvette was flipped upside down onto the padded opening of the
apparatus. The cuvette was rinsed by pouring DI water into the funnel opening of the cleaning
device. The cuvette was rinsed a minimum of three times with water and two times with ethanol.
After each rinse of water or ethanol, the cuvette was shifted slightly to ensure that all liquid was
removed through the vacuum. The cuvette was left on the apparatus for approximately 1 min after
rinsing to completely dry it.
A new glass pipette was used to mix and then transfer the protein samples into the clean
cuvette. Once the cuvette was in place on the spectrometer, the “sample” button on the software
48. 41
window was selected so that the protein sample would be analyzed. When the spectrometer
finished analyzing the sample, a spectrum appeared on the computer screen, and the absorbance at
280 nm was given below the spectrum. The cuvette was again cleaned on the washer/dryer
apparatus. This process was continued until all of the protein samples were tested. The
absorbance values were averaged as needed, and the protein concentration was determined
according to the Beer’s Law Equation (Table 2-15).
Beer’s Law Equation
Equation: A= εlc
Key for Equation
Equation Component Units/Value
A (absorbance at specified peak) No units
ε (Molar absorptivity/ Extinction Coefficient) (Moles/Liter) −1
(centimeters) −1
l ( cell path length/cuvette length) Centimeters
c (concentration of sample) Moles/Liter
SyrB1 Molar absorptivity coefficient: 75,290 M−1
cm−1
[27]
SyrB2 Molar absorptivity coefficient: 59,610 M−1
cm−1
[27]
Table 2-15. Beer’s Law Equation
This equation was used to determine the concentration of the protein solution. The target
concentration was 3-4 mM. If insufficiently concentrated, the protein solution was again
centrifuged in a 10 kDa concentrator filter tube at 3500 g to further reduce the volume and
increase the concentration.
49. 42
Once samples were sufficiently or exhaustively concentrated, the SyrB2 samples were
flash frozen in liquid nitrogen in 100 µL aliquots in Eppendorf tubes. Tubes were then labeled
with concentration, “SyrB2”, and the specific variant. Each tube was then held in the nitrogen
until the protein solution began to freeze and then was released into the liquid nitrogen container
as a hissing noise was heard coming from the tube. An additional 100 µL of protein solution was
extracted using the same pipette tip and expunged into a second Eppendorf tube. Air bubble
release was minimized when either extracting from the centrifuge concentration tube or when
ejecting protein solution into the Eppendorf tubes during the process. The sample freezing process
continued until all of the protein solution was frozen in individual aliquots. The collection of
tubes was stored at -80° C in a Thermo Scientific Forma 900 Series freezer in a freezer-safe box
labeled “SyrB2.”
50. 43
Chapter 3 Results
Chapter 3.1 Cysteine Removal and Cysteine Addition Process
Native cysteine residues at sequence positions 80, 85, 98, and 137 were removed from
wild type SyrB2 and replaced with serine, serine, valine, and alanine, respectively, via “around-
the-horn” mutagenesis. C137 was removed first, and then C98 was removed from the amplified
C137A-encoding plasmid. C80 and C85 were removed from the plasmid containing the C157A
and C98V substitutions in a single round of mutagenesis owing to their close proximity. Four
variants, each containing a single new cysteine residue, were created, starting from the plasmid
encoding the cysteine-free SyrB2. These four variants (N18C, S47C, Q245C, and S258C) were
also created by “around-the-horn” mutagenesis. A native cysteine residue was removed from wild
type SyrB1 at position 419 and replaced with serine. A new cysteine residue was added to
position 582 in exchange for threonine to create a C419S, T582C double variant construct. The
success of the polymerase chain reaction cycle allowed new variants to be created and is
evidenced by the presence of bands on an agarose gel after electrophoresis (Figure 3-1). Because
no DNA product was added to the PCR mix in a high enough concentration to have appeared on
the gel, the presence of bands verified that the PCR reaction was successful in amplifying a
double-stranded DNA of similar size.
51. 44
Figure 3-1. Ethidium Bromide Stained Agarose Gel of PCR Reactions Visualized under UV
Light
An agarose gel containing samples of unligated PCR product under ultraviolet light.
The bottom row is the DNA ladder (blue); 1:10 diluted template plasmid concentration samples
(white text); 1:100 diluted template plasmid concentration samples (black text); number is the
annealing temperature used for that sample
Chapter 3.2 Results of Initial Protein Purifications and UV/VIS Absorption Analysis
SDS-PAGE gels indicated that SyrB2 S258C variants were expressing but may perhaps
not survive the purification process (Figure 3-2). Lane 3 in this figure is noticeably bluer due to
the higher concentration of purified, but likely contaminated, product. The gel does not show a
distinctive band at the expected location for the SyrB2 protein. The presence of contaminants
apparent on this gel was alarming, but the greater issue was the poorly-expressed SyrB2 variant.
67° C
69° C
71° C
65° C
67° C
69° C
71° C
DNA Ladder
52. 45
Figure 3-2. SDS-PAGE Gel after Initial SyrB2 S258C Purification
The gel above shows analysis of a sample resulting from purification of the S258C variant of
SyrB2. Protein ladder (Lane 1); protein sample directly after elution (Lane 2); protein sample
after concentration by centrifugation (Lane 3)
Absorption spectroscopy was used to quantify the SyrB2 that may have been present in
the sample. Spectral results confirmed doubts about the purity of the SyrB2 S258C protein that
was eluted (Figure 3-4). A peak at 280nm was expected, as indicated by the vertical red line.
However, the only peak that appears is at 260 nm, indicating a significantly larger amount of
imidazole, nucleic acids, or other contaminants such as nucleic acids. Because this result
suggested a potential problem with the S258C variant, the SyrB2 Q245C variant was used for
subsequent expression and purification tests. In principle, this variant might be more easily
expressed and purified.
1
2
3
53. 46
Figure 3-3. UV/VIS Results for SyrB2 S258C
The figure shows the absorption spectra of a sample of the protein with a lower concentration
(1:400 sample to buffer instead of 1:100 as used with the three higher peaks) (light blue peak).
The plasmid encoding the SyrB2 Q245C variant was used to transform competent E. coli
BL21(DE3) cells, and the Q245C variant protein was expressed by the procedure given in
Chapter 2.9. Samples were taken from the discarded loading and column wash eluate, and these
were analyzed on and SDS-PAGE gel (Figure 3-4). Dark bands appeared at the expected location
for SyrB2 in both discarded solutions, indicating poor solubility of the protein.
54. 47
Figure 3-4. SDS PAGE Gel Showing SyrB2 Q245C Variant Protein in Loading and Column-wash
Eluates (High Contrast)
The SyrB2 Q245C variant appears not to readily adsorb to the Ni(II)-NTA agarose column and
appears in the loading (Lane 2) and wash (Lane 3) eluates. The arrows mark the expected location
of SyrB2. There was a darker and thicker band at this location, indicating the presence of the
protein in the column-loading eluate.
The results from the initial SyrB2 S258C and Q245C variant expression and purification
tests suggested that the new SyrB2 variant might be challenging to express in soluble form. To
address this issue, a set of parameters was listed to create a series of expression tests to improve
soluble, stable protein yield. The critical parameters were determined to be cell density (as
reported by OD600) at induction, concentration of IPTG used to induce, time between induction
and harvest, and elution/equilibration buffer ingredients. The full list of expression tests was
described in Chapter 2.10.b., and the results of modifying several experimental parameters on
protein production in three expression tests is described in the next section.
55. 48
Chapter 3.3 Expression Test Results
In the first round of expression tests, all critical parameters were considered as possible
reasons for low protein yield. Cell density at the time of induction, concentration of IPTG, and
time between induction and cell harvest were all varied in the first expression test. Cells were
induced at OD600 values of either 0.3-0.4 or 0.6-0.8. IPTG used for induction was 25, 125, or 503
µM. Time after induction was either 4 or 22 hours. Each of the twelve flasks used for this analysis
had at least one variable different than any other flask.
After induction, the yield of expression of SyrB2 S258C protein was qualitatively
determined by analyzing seven selected solutions containing lysed cells on SDS-PAGE (Table 3-
1 and Figure 3-5). In addition to these seven samples, aliquots of cell-solution that had not been
induced were also included to determine if induction affected protein solubility. Gel samples from
the first expression test showed that the yield of SyrB2 variant expression varied with the
concentration of IPTG and pre-induction cell density.
Lane 1 2 3 4 5 6 7 8 9 10
Absorbance N/A;
Protein
Ladder
Lane
0.3-
0.4
0.6-
0.8
0.3-
0.4
0.6-
0.8
0.3-
0.4
0.6-
0.8
0.3-
0.4
0.6-
0.8
0.3-
0.4
Amount of
IPTG
Pre-
IPTG
Pre-
IPTG
25 µM 125
µM
503
µM
503
µM
25 µM 125
µM
125
µM
Time of
Sample
0 0 +4
Hours
+4
Hours
+4
Hours
+4
Hours
+ 22
Hours
+ 22
Hours
+ 22
Hours
Table 3-1. Expression Test Gel 1 Key
The table above shows the parameters for each sample in SDS-PAGE (Figure 3-5). Lanes that
strongly expressed SyrB2 S258C are highlighted in green. Lanes that may have weakly expressed
56. 49
the SyrB2 variant are highlighted yellow. Several samples were omitted due to the limited
number of gel lanes. The samples omitted were not thought to be significantly different from the
samples used.
Figure 3-5. Expression Test 1 Gel (High Contrast)
The figure above shows a highly contrasted image of the SDS PAGE gel for expression test 1.
The contrast was used to highlight the band suspected to be the SyrB2 variant. Arrows are
indicate the dark bands in lanes 2, 3, 6, and 9 that likely indicate the presence of the S258C
variant SyrB2.
As depicted (Table 3-1), the SyrB2 S258C variant was expressed in several samples.
Lanes 2, 3, 6, and 9 show high levels of expression of SyrB2 S258C under these conditions.
Furthermore, lanes 7 and 8 appear to show modest expression, indicating successful expression
across the parameter space. Pre-IPTG samples were expected to contain the protein as well, and
confirmed the ability of the competent E. coli to produce the SyrB2 mutant without chemical
induction. These results were encouraging for optimizing parameters in a second round of
expression tests, which is described next.
57. 50
Several additional changes were made in the second round of expression tests to try to
improve upon the successful first experiment. Cell density was determined to be irrelevant to the
success of protein expression, given that SyrB2 was overexpressed in flasks induced at both
OD600 = 0.3-0.4 and OD600 = 0.6-0.8. IPTG was again varied at 25, 125, and 503 µM as the first
round of expression tests were inconclusive regarding the effects of IPTG on protein yield. A
wider range of post-induction, pre-harvest interval was used in this experiment because the
protein was adequately concentrated in both 4 h and 22 h tests. A wider time range was expected
to contribute to finding a more optimal (if not only for efficiency purposes) interval. Samples
were taken for SDS-PAGE analysis from eight of the twelve total cultures grown, and results are
depicted below (Table 3-2, Figure 3-6).
Lane 1 2 3 4 5 6 7 8 9 10
Absorbance N/A;
Protein
0.4-0.5 0.4-
0.5
0.4-0.5 0.4-
0.5
0.4-
0.5
0.4-0.5 0.4-
0.5
0.4-
0.5
0.4-0.5
Amount of
IPTG
Ladder
Lane
Pre-
IPTG;
chilled
shocked
cells
25 µM 503
µM
25
µM
125
µM
503
µM
25
µM
125
µM
503
µM
Time of
Sample
2:30 pm 6pm 6pm 7:30
pm
7:30
pm
7:30
pm
10:30
am
10:30
am
10:30
am
Table 3-2. Expression Test 2 Gel Key
58. 51
The table above shows the variables used for each sample analyzed by SDS-PAGE (Figure 3-6).
Conditions revealed by gel to yield a high level of expression of SyrB2 S258C are highlighted in
green. Condition leading to weak but detectable expression are highlighted yellow.
Figure 3-6. Expression Test 2 Gel Result
The above image shows the gel result from expression test 2. Again, several samples were
omitted; these samples were expected not to differ significantly from the samples selected for
analysis. Lanes 5, 8, and 9 appear to reveal significant expression of SyrB2 S258C, as indicated
by arrows pointing to the lower dark bands in each of these lanes.
As indicated on the gel, lanes 5, 8, and 9 reveal a high level of expression of the SyrB2
S258C variant. Lane 10 also appears to show a low level of expression. Experimentally, this
result, in combination with the first expression test, raised more questions than it definitively
answered. On a positive note, 125 µM IPTG and 22 h of post-induction, pre-harvest time proved
to allow for robust SyrB2 production in both expression tests. However, discrepancies existed
between the first and second expression tests on the optimal concentration of IPTG needed to
effectively induce overexpression. In the first test, 503 µM appeared best. In the second test, the
59. 52
samples containing a 25 µM concentation of IPTG appeared to be more effective. Together, the
results were inconclusive. There was also a possibility that the variation in the concentration of
IPTG from was insignificant and thus the success of expression was independent of this factor.
Additionally, in the second expression tests, flasks were chilled prior to induction. This variable
likely negatively affected expression levels. In the first expression test, robust levels of protein
were expressed in the pre-IPTG samples, yet, in the chilled pre-IPTG samples in test two, there
was no detectable protein expression. This result suggests that the SyrB2 variant might be
temperature sensitive. However, this result was not taken as certain, and the cells were again
chilled in the third expression test experiment.
Because the 6mg IPTG samples showed overpression of SyrB2 at multiple post-induction
time points (Table 3-2), these cells were expected to have the greatest chance of successful
purification. Given previous concerns over protein stability during purification, this protein was
purified using HEPES elution and equilibration buffers that contained glycerol and KCl. Samples
of the cell pellet, column loading eluate, and as-eluted protein samples were mixed with loading
dye, boiled for four min, and analyzed on an SDS-PAGE gel. The results of this gel are shown
below (Figure 3-7).
60. 53
Figure 3-7. 6 mg IPTG Induced, SyrB2 S258C Expressing E. Coli Pellet and Purification Gel
Protein Ladder (1); Cell paste (2); Column Flowthrough (3); Eluted Protein (4)
The resulting gel from the 6mg IPTG SyrB2 S258C purification is shown above. It was
hoped that a large band would appear around 35 kDa in the eluted protein lane. Unfortunately, no
major bands appear in the elution lane at the expected position for the SyrB2 variant. Both the
cell pellet and the flowthrough from the column contain a mix of many different proteins. While,
again, no distinct band can be seen at the expected 35 kDa mark on either of these lanes, the dark
coloration within the 35 kDa region indicates that SyrB2 was likely washed out during the elution
process. At this point, the purification procedure needed to be revisited. More protein samples
had to be expressed in E. coli, thus presenting the additional opportunities to further optimize
expression and "kill two birds with one stone."
The third expression test again varied IPTG concentration used for induction and interval
between induction and harvest. In this experiment, induction was stopped when the sample was
taken. For example, when the 8pm samples were removed, the flasks from which these samples
61. 54
were taken were removed from the incubator, and the cells were centrifuged, frozen in liquid
nitrogen, and stored at -80° C. This differed from the past experiments, when the cells were left to
incubate after samples were taken at the designated time periods. On the basis of the success of
previous experiments with lesser concentrations of IPTG previously, only 25 µM and 83 µM was
used. Seven cell solutions containing concentrations of either 25 µM or 83 µM of IPTG at various
time points were selected for SDS-PAGE analysis (Table 3-3, Figure 3-8). In this experiment,
longer time intervals between induction and harvest proved to be the most beneficial to SyrB2
protein expression.
Lane 1 2 3 4 5 6 7 8 9
Absorbance N/A;
Protein
Ladder
Lane
0.4-
0.5
0.4-
0.5
0.4-0.5 0.4-
0.5
0.4-
0.5
0.4-
0.5
0.4-
0.5
0.4-
0.5
Amount of
IPTG
Pre-
IPTG
83 µM 25 µM 83
µM
25
µM
83 µM 25
µM
83
µM
Time of
Sample
12 pm 4:30
pm
6:30pm 6:30
pm
8 pm 8 pm 9 am 9 am
Table 3-3. Expression Test 3 Gel Key
The table above gives the variables for samples on the SDS-PAGE gel (Figure 3-8). Lanes that
show strong expression of the SyrB2 S258C variant are highlighted in green. Lanes that may
show weak expression are highlighted yellow. Some samples taken in the afternoon of day 1 were
omitted.
62. 55
Figure 3-8. Expression Test 3 Gel
The above image shows the gel used to optimize conditions for SyrB2 S258C expression in the
third expression test. Potential SyrB2 S258C bands are shown with arrows.
The results of the third expression test confirm that, despite some examples of outliers,
more time after induction is better for protein overexpression. This parameter is critical to
understanding the nature of the SyrB2 variants produced, as it had been hypothesized that too
much protein may lead to aggregation of insoluble clumps (inclusion bodies) within the cell
pellet. Additionally, rapid chilling of the cells almost certainly had a negative effect on protein
production. Pre-IPTG cells that had been chilled again did not show any detectable SyrB2
expression, and longer post-induction periods may have produced the most protein yield due to
the initially deleterious temperature effect on protein structure and resulting solubility.
Following this experiment-altering realization, cells that were incubated with either 25
µM or 83 µM of IPTG until 8pm (and were then centrifuged to remove media, frozen, and stored
as previously described) were combined and purified as a negative control. Given that previous
63. 56
expression of cells around this time period in expression test 2 with this amount of IPTG had
shown SyrB2, this purification would confirm if any protein could be extracted from this time
sample. Results of this purification are shown below (Figure 3-9). As expected in this negative
control test, no protein was seen in the eluted protein sample, confirming the results of the post-
induction gel. From this analysis, it can be determined that longer post-induction incubation time
(likely more than 20 hours), lesser concentrations of IPTG (25-83 µM), and no chilling optimized
expression.
Figure 3-9. Purification of SyrB2 S258C from Third Expression Test
The gel above shows the results of purifying protein from Expression Test 3. From 1-5, the lanes
show: the protein ladder, the cell pellet, loading eluate, column wash eluate, eluted protein
sample, respectively. As indicated by arrows, the cell pellet and column loading eluate samples
have a large amount of the SyrB2 variant.
64. 57
Chapter 4 Discussion and Direction of Future Research
Chapter 4.1 "Around-the-Horn" Mutagenesis
Although some initial complications with polymerases were encountered, a successful
process was designed for cysteine-removal and cysteine-addition mutagenesis. The phusion
polymerase was remarkably more effective than either taq or Q5 polymerase in bringing about the
desired mutagenesis. The ineffectiveness of taq and Q5 may have been due to the enzymes having
been improperly stored or expired; however, there may be an explanation based on the limitations
of the polymerases. For one, Taq polymerase may not have been able to amplify the large, 6000
base pair plasmid, as it is typically used for plasmids with less than 5,000 base pairs [30].
Additionally, Taq does not proofread the sequence it amplifies, leading to errors at a rate of
4.5x10-5
errors/base pair and may have not been able to amplify the template DNA strand because
of the occurrence of unwanted substitutions [31]. The phusion polymerase was thus used to create
all variant constructs and did so with a very high success rate. The phusion polymerase is
considered a high-fidelity polymerase and proofreads the DNA sequence. It has an error rate of 2-
3x10-6
per base pair, a value more than twenty times less than the error rate of Taq and much
more beneficial for controlled mutagenesis. On the other hand, Q5 polymerase is also a high-
fidelity polymerase, but may have been used with too short of an extension time (this possibility
was suggested to us by Dr. Benjamin Allen).
Additionally, the DNA extraction protocol was altered to improve upon low yields in
early mutagenesis experiments. The increases in buffers A1, A2, and A3 led to an increased
effectiveness of cell resuspension, lysis, and neutralization, respectively. When mixing buffers
through tube inversion, samples needed to be uniform to maximize that buffer’s effect on the
65. 58
entire cell sample. Buffer AW helped to wash away additional contaminants to safeguard against
impurities. The drying time was increased to ensure that only plasmid remained on the filter
membrane before elution. Buffer AE was found to be effective for elution without heating to 70°
C. After the initial elution buffer flowed through the column membrane due to centrifugation at
11,000 g for 1 min, the solution was added back onto the column. The tube was then centrifuged
at 11,000 g for 2 min. Double elution helped to solubilize any of the DNA that may have
remained on the column after the first elution due to the small amount of Buffer AE used (50 µL).
These changes helped to increase DNA concentration yield to as much as 500 ng/µL, which
represented a significant improvement from values often less than 100 ng/µL in early
experiments. For reference, a concentration of at least 200 ng/µL is recommended for DNA
sequencing. DNA samples that were extracted with the modified procedure therefore did not
require additional SpeedVac concentration. Thanks to the modified DNA extraction procedure, all
intended SyrB1 and SyrB2 mutant plasmids were created in abundant concentrations.
Chapter 4.2 Initial SyrB2 Protein Expression and UV/VIS Result Analysis
The first attempt at expressing and purifying SyrB2 S258C appeared to be unsuccessful
based on a lack of clear protein bands at the 35 KDa marker on the expression protein gel shown
in Figure 3-2. No distinguishable band can be seen on this gel, which could indicate one of two
things. First, the protein might not have been expressed in E. coli or, second, the protein might not
have folded correctly or might have been lost during the purification for some other reason.
However, the presence of loosely defined, impure bands that seemed to form a smear of blue
color on the gel were puzzling, and so further purification and dialysis of the sample was
performed to determine if a small amount of SyrB2 was still present. The absorption spectra
shown in Figure 3-4 gave the result of the additional purification and dialysis effort and
66. 59
confirmed that no SyrB2 was present in the sample. The 260 nm peak shown in the spectrum is
attributable to nucleotide or nucleic acid contaminants as well as imidazole, a chemical
component of the equilibration and elution buffers. The amino acids, tryptophan and tyrosine,
both present in the protein sequences, have absorption peaks in the 280 nm region and were
expected to confirm the presence of SyrB2. It appears that only contaminants, and no SyrB2
protein, were present in this sample based on these spectral results.
Figure 3-3 provides some limited insight as to when the SyrB2 variants were being lost in
the process of expression and purification. The presence of thick, distinct bands around 35 kDa on
the gel in the lanes containing the column loading and column wash eluates suggests that the
protein may have been expressed but may not have adsorbed to the column. However, the number
of other contaminants also present on this gel makes this conclusion uncertain. Therefore, protein
expression tests were needed to further investigate this hypothesis.
Chapter 4.3 Expression Test Discussion
The first round of expression tests was done to determine if SyrB2 S258C could be
expressed under some conditions in E. coli BL21(DE3) cells. As demonstrated in Figure 3-5, the
test confirmed that the variant was expressed under a range of expression conditions shown on
the gel and described in Table 3-1. It was determined from this set of tests that, because samples
that had been induced at cell densities corresponding to OD600 0.3-0.4 and 0.6-0.8, cell density
was not critical to protein expression. Therefore, in subsequent experiments, samples were
induced at OD600 = 0.4-0.5, as this range provided a robust mid-range. The cell samples from this
expression test were collected and stored as previously described. Interestingly, only a yield of
cells was produced in each case, possibly indicating that the cells were more involved with
protein expression than with cell multiplication.