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.
Calcium (Ca2+) binding is essential for the structure and activity of complex I in Escherichia coli. Purified complex I is strongly inhibited by the calcium chelator EDTA, with half maximal inhibition at 2.5 μM EDTA. Membrane-bound complex I is less sensitive to EDTA inhibition than purified complex I, indicating calcium binding is affected by removal from the membrane. Reconstituted complex I has intermediate sensitivity to EDTA, becoming as sensitive as purified complex I after washing, suggesting a loss of factors that influence calcium binding upon purification and reconstitution. Tight binding of calcium is required to maintain complex I activity.
Determination of 8-Hydroxy-2 Deoxyguanosine in Pseudomonas Fluorescens Freeze...Agriculture Journal IJOEAR
Abstract— Oxidative DNA damage is involved in the f cell death induced by freeze-dried powder during storage. Cell 8-hydroxy-2’deoxyguanosine (8-oxodG) is widely accepted as a biomarker of the “freeze-dried bacteria” oxidative DNA damage. The aim of this study was to introduce a method for determination 8-oxodG in cell freeze-dried samples using high-performance liquid chromatography with electrochemical detection. In the tested range of 0.5 µmol L-1 to 1.0 nmol L-1, the calibration curve was linear (r2=0.9995) and the limit of detection was 0.05 µmol L-1. The used method did not allow highlighting the presence in the samples of the 8OH within the limits of detection. A more successful method (more sensitive) would be needed to detect possibly the 8OH.
His Tag Protein Production and PurificationExpedeon
The study of protein regulation, structure, and function relies heavily on the expression and purification of recombinant proteins. Many recombinant proteins are expressed as fusion proteins, meaning that they contain an affinity / epitope tag. A tag is a short sequence of DNA that codes for a specific amino acid, which is frequently inserted into a target gene at the point of coding for expression at either the N or C terminal of the protein required.
Novel creation of 3-contiguous stereocenters via a patented an asymmetric catalytic process utilizing Titanium and Zinc, in conjunction with either oxygen or peroxides.
IRJET- Understanding the cDNA isolation and antimitogenic property in plant l...IRJET Journal
This document summarizes research on the isolation and characterization of cDNA from plant lectins and their anti-mitogenic properties. Key points include:
- Plant lectins are proteins found in many plants that bind carbohydrates and can agglutinate red blood cells. Their structure gives therapeutic and biotechnological potential.
- Researchers have isolated cDNA from various plants like Glechoma hederacea and Salvia stenophylla to study lectin genes and properties. Methods included RNA isolation, cDNA library construction, sequencing, and molecular modeling.
- Studies found that isolated lectins showed anti-mitogenic effects, inhibiting the growth and killing of some cancer cell lines, utilizing their carbohydrate-binding and toxic
This document summarizes research on the effects of heme ring oxygenation on the structure and function of cytochrome c peroxidase (CcP). Specifically, it describes the synthesis of 4-mesoporphyrinone (mesopone) and its incorporation into CcP to form a hybrid protein called MpCcP. Testing found that MpCcP had similar peroxidase activity to wild-type CcP with cytochrome c, but varied activity with other substrates. Structural analysis via X-ray crystallography provided the first structural characterization of an oxygenated heme protein and found only the S-isomer of mesopone in the crystallized protein despite using a mixture of isomers.
Calcium (Ca2+) binding is essential for the structure and activity of complex I in Escherichia coli. Purified complex I is strongly inhibited by the calcium chelator EDTA, with half maximal inhibition at 2.5 μM EDTA. Membrane-bound complex I is less sensitive to EDTA inhibition than purified complex I, indicating calcium binding is affected by removal from the membrane. Reconstituted complex I has intermediate sensitivity to EDTA, becoming as sensitive as purified complex I after washing, suggesting a loss of factors that influence calcium binding upon purification and reconstitution. Tight binding of calcium is required to maintain complex I activity.
Determination of 8-Hydroxy-2 Deoxyguanosine in Pseudomonas Fluorescens Freeze...Agriculture Journal IJOEAR
Abstract— Oxidative DNA damage is involved in the f cell death induced by freeze-dried powder during storage. Cell 8-hydroxy-2’deoxyguanosine (8-oxodG) is widely accepted as a biomarker of the “freeze-dried bacteria” oxidative DNA damage. The aim of this study was to introduce a method for determination 8-oxodG in cell freeze-dried samples using high-performance liquid chromatography with electrochemical detection. In the tested range of 0.5 µmol L-1 to 1.0 nmol L-1, the calibration curve was linear (r2=0.9995) and the limit of detection was 0.05 µmol L-1. The used method did not allow highlighting the presence in the samples of the 8OH within the limits of detection. A more successful method (more sensitive) would be needed to detect possibly the 8OH.
His Tag Protein Production and PurificationExpedeon
The study of protein regulation, structure, and function relies heavily on the expression and purification of recombinant proteins. Many recombinant proteins are expressed as fusion proteins, meaning that they contain an affinity / epitope tag. A tag is a short sequence of DNA that codes for a specific amino acid, which is frequently inserted into a target gene at the point of coding for expression at either the N or C terminal of the protein required.
Novel creation of 3-contiguous stereocenters via a patented an asymmetric catalytic process utilizing Titanium and Zinc, in conjunction with either oxygen or peroxides.
IRJET- Understanding the cDNA isolation and antimitogenic property in plant l...IRJET Journal
This document summarizes research on the isolation and characterization of cDNA from plant lectins and their anti-mitogenic properties. Key points include:
- Plant lectins are proteins found in many plants that bind carbohydrates and can agglutinate red blood cells. Their structure gives therapeutic and biotechnological potential.
- Researchers have isolated cDNA from various plants like Glechoma hederacea and Salvia stenophylla to study lectin genes and properties. Methods included RNA isolation, cDNA library construction, sequencing, and molecular modeling.
- Studies found that isolated lectins showed anti-mitogenic effects, inhibiting the growth and killing of some cancer cell lines, utilizing their carbohydrate-binding and toxic
This document summarizes research on the effects of heme ring oxygenation on the structure and function of cytochrome c peroxidase (CcP). Specifically, it describes the synthesis of 4-mesoporphyrinone (mesopone) and its incorporation into CcP to form a hybrid protein called MpCcP. Testing found that MpCcP had similar peroxidase activity to wild-type CcP with cytochrome c, but varied activity with other substrates. Structural analysis via X-ray crystallography provided the first structural characterization of an oxygenated heme protein and found only the S-isomer of mesopone in the crystallized protein despite using a mixture of isomers.
This document summarizes research on immobilizing palladium nanoparticles with a functional ionic liquid grafted onto a cross-linked polymer for solvent-free Heck reactions. Specifically:
1) A functional ionic liquid containing an amine group was synthesized and copolymerized with divinylbenzene to produce a cross-linked polymer support.
2) Palladium nanoparticles were immobilized on this polymer using aqueous palladium chloride, then reduced with sodium borohydride.
3) Characterization with FTIR, TGA, TEM, XPS showed the palladium nanoparticles were successfully supported on the polymer through amine binding.
4) This catalyst was tested for Heck
This document describes research on triplex formation by oligodeoxynucleotides (ODNs) containing 5-methyldeoxycytidine conjugated to spermine (5-Me-dC-N4-(spermine)). The key findings are:
1) ODNs containing 5-Me-dC-N4-(spermine) form stable triplexes at physiological pH (pH 7.3), unlike unmodified ODNs which only form triplexes under acidic conditions.
2) The triplex stability for 5-Me-dC-N4-(spermine) ODNs decreases with decreasing pH, in contrast to unmodified ODNs whose stability increases under acidic conditions
The document describes the synthesis and evaluation of 6,7-dihydro-4H-isothiazolo[4,5-b]pyridin-5-ones (DIP) as potential antimitotic agents. A series of DIP derivatives were synthesized via a multicomponent reaction. Selected DIP derivatives were found to alter sea urchin egg cleavage at low nanomolar concentrations and showed cytotoxicity against cancer cells, including chemoresistant lines, at submicromolar to low nanomolar levels. Both the sea urchin embryo assay and cancer cell assays confirmed the DIP derivatives act by destabilizing microtubules. Structure-activity relationship studies showed the importance of a p
1) The document describes a novel method called INLIGHTTM for the relative quantification of N-linked glycans using isotopically labeled glycan hydrazide tags.
2) The method involves releasing N-linked glycans from glycoproteins using PNGase F, then derivatizing the glycans from two samples with either a light or heavy tagged reagent.
3) The tagged glycans are mixed, analyzed by LC/MS, and ratios of light and heavy glycan pairs are calculated to quantify differences between the two samples after correcting for isotopic overlap.
This document presents the results of a study aiming to develop selective inhibitors of BACE2 using computer-aided drug design. BACE2 is homologous to BACE1 but is predominantly expressed in peripheral tissues and may serve as an alternative protease in APP processing. The study validated docking programs for reproducing binding poses of BACE1 and BACE2 inhibitors and evaluated scoring functions for virtual screening. A BACE2 homology model produced better virtual screening results than crystal structures. Future work will involve virtual screening larger libraries and designing new scaffolds for selective BACE2 inhibition.
The document discusses protein purification techniques used in research and industry. It describes a protein expression and purification series that teaches students to express and purify the protein dihydrofolate reductase (DHFR) using different modules, including bacterial cell culture, affinity chromatography using nickel beads, and size exclusion chromatography to remove imidazole. The series mimics real-world protein purification workflows and allows students to purify a non-colored protein and analyze it using techniques like SDS-PAGE electrophoresis and enzymatic assays.
Enhanced electrophoretic resolution of monosulfate GAG disacchride isomers on...Yong Zhang
This document describes research on improving the separation of monosulfate glycosaminoglycan disaccharide isomers by microchip electrophoresis. Key findings include:
1) Addition of 1,4-dioxane (DO) to the running buffer dramatically improved resolution of the isomers, likely due to solvation effects.
2) Methylcellulose was used to suppress electroosmotic flow and analyte adsorption to the poly(methyl methacrylate) microchip surface.
3) Optimization of buffer pH, addition of beta-cyclodextrin, and concentration of 1,4-dioxane enhanced resolution of the monosulfate isomers.
4) Under optimized conditions,
This document summarizes research into the HemQ enzyme from Staphylococcus aureus, which catalyzes the final step in the biosynthesis of heme b in some bacteria. The researchers sought to identify reaction intermediates on the pathway from coproheme III to heme b. They found that the first decarboxylation rapidly produces harderoheme III as an intermediate, while the second decarboxylation controls the overall rate. Both harderoheme isomers III and IV reacted when bound to HemQ. While H2O2 is the presumed biological oxidant, peracetic acid could also drive the reaction, suggesting possible iron-mediated reaction mechanisms.
1) Rv3717 is a novel N-acetylmuramoyl-L-alanine amidase enzyme from Mycobacterium tuberculosis that was determined to have a single-domain structure at 1.7 Angstrom resolution.
2) Unlike other bacterial autolysins, Rv3717 lacks a separate cell-wall binding domain and instead uses its net positive charge for substrate binding.
3) The crystal structure revealed a flexible hairpin turn that partially occludes the active site, which may be involved in autoregulation of enzymatic activity similar to other bacterial amidases.
2D gel electrophoresis separates proteins based on their isoelectric point (pI) in the first dimension and molecular weight in the second dimension. Amino acids can exist as zwitterions and act as both acids and bases, donating or accepting protons. The isoelectric point is calculated using the Henderson-Hasselbalch equation and titration curves, taking into account the pKa values of amino acid side chains. Detection methods for proteins on 2D gels include staining with dyes or radioactive labeling.
Chromium-induced growth inhibition and alteration of biochemical parameters i...ijtsrd
The hydroponically grown plants of Ocimum basilicum L. were exposed to varying levels of K2Cr2O7 (0, 5, 10, 25 -µM). The plants were tested for various morphological and biochemical parameters on 3rd and 5th day after treatment. Chromium (Cr) resulted in reduction of plant length and biomass. The deleterious effects of the hexavalent chromium on O. basilicum were further confirmed by the reductions in chlorophyll a and b contents, soluble protein and while as the free amino acid and proline contents were increased. The study concludes that chromium causes stress in the Ocimum basilicum plants and thus alters various morphological and biochemical parameters. Ruqaya Jabeen"Chromium-induced growth inhibition and alteration of biochemical parameters in Ocimum basilicum L." Published in International Journal of Trend in Scientific Research and Development (ijtsrd), ISSN: 2456-6470, Volume-2 | Issue-2 , February 2018, URL: http://www.ijtsrd.com/papers/ijtsrd8329.pdf http://www.ijtsrd.com/chemistry/other/8329/chromium-induced-growth-inhibition-and-alteration-of--biochemical-parameters-in-ocimum-basilicum-l/ruqaya-jabeen
This document reports on the synthesis and characterization of polyethyleneimine-anchored copper(II) complexes and their in vitro DNA binding studies and cytotoxicity. Specifically, it synthesized copper(II) complexes containing 1,10-phenanthroline and L-tyrosine ligands bound to a branched polyethyleneimine polymer. It characterized the complexes using various techniques and studied their binding to calf thymus DNA. It found that the complex with the highest degree of copper(II) coordination bound most strongly to DNA. Finally, it evaluated the cytotoxic activity of this complex against MCF-7 breast cancer cells.
This document describes a new capillary zone electrophoresis (CZE) method for the simultaneous quantitative determination of β-N-oxalyl-L-α,β-diaminopropionic acid (β-ODAP) and homoarginine in Lathyrus sativus (grass pea). The method uses a new sodium borate-sodium sulfate run buffer at pH 9.2. It was found that the ratio of α- and β-ODAP isomers changes from 34.5/65.5 to 28.6/71.4 as the pH increases from 3.0 to 11.0. The method allows for the fast, simple, and sensitive determination of β-OD
This study assessed the neurotoxicity of cobalt (Co) on SH-SY5Y human neuroblastoma cells in vitro. The results showed that CoCl2 reduced cell viability in a dose-dependent manner when measured by MTT and NR assays at 24, 48, and 72 hours. CoCl2 treatment caused cell shrinkage and apoptosis. Antioxidants provided some protection against Co toxicity at lower concentrations but not at higher concentrations, suggesting oxidative stress may not be the main mechanism of toxicity. Overall, the results indicate that Co is toxic to neurons and this toxicity could relate to neurological symptoms reported in patients with cobalt-chromium hip implants who have elevated blood Co levels.
Nucleoside analogues synthesis using natural phosphate doped with i2 (npi2) i...Alexander Decker
This document summarizes research on the synthesis of nucleoside analogues using natural phosphate doped with iodine (NP/I2) as a catalyst. Several D-ribonucleosides were prepared from 1-O-acetyl-2,3,5-tri-O-benzoyl-β-D-ribofuranoside and silylated nucleobases under mild conditions using NP/I2. Yields of the desired nucleosides ranged from 35-62% depending on the nucleobase and amount of iodine used. NP/I2 was shown to be a more effective solid catalyst than silica or alumina doped with iodine. The reaction is proposed to
Proteinase K is a serine protease isolated from a fungus that is able to digest keratin. It has broad substrate specificity and can degrade many native proteins even in the presence of detergents. Proteinase K is commonly used in molecular biology to digest unwanted proteins from nucleic acid preparations. It works best at pH 7.5-8.0 and 37°C, and is typically used at concentrations of 50-200 μg/ml for 30 minutes to 18 hours. Proteinase K is stable and active over a broad range of conditions.
This document summarizes the total synthesis of 2,6-dideoxy-2,6-imino-7-O-β-D-glucopyranosyl-D-glycero-L-gulo-heptitol hydrochloride (8), a potent inhibitor of α-glucosidases. The key steps involve homologation and amination of 2,3,4,6-tetra-O-benzyl-D-glucopyranose (1) to form the protected amine 4. An intramolecular cyclization of 4 catalyzed by mercuric acetate formed the piperidine ring. Glycosylation of aglycon 6 with acetob
This document describes a study characterizing a novel gene cluster involved in the degradation of 4-chlorocatechol by Pseudomonas reinekei MT1. The researchers found that during growth on 5-chlorosalicylate, a novel (chloro)catechol 1,2-dioxygenase (C12OccaA) and a novel (chloro)muconate cycloisomerase (MCIccaB) were induced. MCIccaB was found to transform 3-chloromuconate into equal amounts of cis-dienelactone and protoanemonin, acting as a functional intermediate between known chloromuconate cycloisomerases and muconate cycloisomerases
ABSTRACT- L-Ascorbic acid derivatives was synthesized on treatment with acetone and acetyl chloride afforded 5,6-acetal of L-ascorbic acid then benzylation of C-2 and C-3 hydroxyl groups of the lactone ring was accomplished using K2CO3 and benzyl bromide in DMF, then deblocking of the 5,6-O,O-protected derivative of L-Ascorbic acid with acetic acid and methanol gave 2,3-O,O-dibenzyl-L-Ascorbic acid. Subsequently mono-tosylation at 6 position of 2,3-O, O-dibenzyl-L-Ascorbic acid was carried out with addition of p-toluenetosylchloride (PTSC) in Pyridine and MDC solvent medium gave 2,3-O,O-dibenzyl-6-O-tosyl-L-Ascorbic acid. All the structures were characterized by 1H NMR, 13C NMR and Mass Spectroscopy.
Key-words- L-Ascorbic acid, 5,6-Acetal, Benzylation, Hydrolysis, Tosylation
1. Researchers developed a Si-tag fusion protein containing Si-tag, RGD, and His-tag to enable delivery of biomolecules like RGD onto silica surfaces for tissue engineering applications.
2. They expressed the fusion protein in E. coli and tested various purification methods, finding that rapid purification using silica nanoparticles was more effective than silica gel or IMAC.
3. Functionality assays using immunostaining and binding to silica nanoparticles confirmed the fusion protein retained its ability to bind silica surfaces. Further optimization of purification conditions is still needed to improve protein yield.
This document summarizes research on immobilizing palladium nanoparticles with a functional ionic liquid grafted onto a cross-linked polymer for solvent-free Heck reactions. Specifically:
1) A functional ionic liquid containing an amine group was synthesized and copolymerized with divinylbenzene to produce a cross-linked polymer support.
2) Palladium nanoparticles were immobilized on this polymer using aqueous palladium chloride, then reduced with sodium borohydride.
3) Characterization with FTIR, TGA, TEM, XPS showed the palladium nanoparticles were successfully supported on the polymer through amine binding.
4) This catalyst was tested for Heck
This document describes research on triplex formation by oligodeoxynucleotides (ODNs) containing 5-methyldeoxycytidine conjugated to spermine (5-Me-dC-N4-(spermine)). The key findings are:
1) ODNs containing 5-Me-dC-N4-(spermine) form stable triplexes at physiological pH (pH 7.3), unlike unmodified ODNs which only form triplexes under acidic conditions.
2) The triplex stability for 5-Me-dC-N4-(spermine) ODNs decreases with decreasing pH, in contrast to unmodified ODNs whose stability increases under acidic conditions
The document describes the synthesis and evaluation of 6,7-dihydro-4H-isothiazolo[4,5-b]pyridin-5-ones (DIP) as potential antimitotic agents. A series of DIP derivatives were synthesized via a multicomponent reaction. Selected DIP derivatives were found to alter sea urchin egg cleavage at low nanomolar concentrations and showed cytotoxicity against cancer cells, including chemoresistant lines, at submicromolar to low nanomolar levels. Both the sea urchin embryo assay and cancer cell assays confirmed the DIP derivatives act by destabilizing microtubules. Structure-activity relationship studies showed the importance of a p
1) The document describes a novel method called INLIGHTTM for the relative quantification of N-linked glycans using isotopically labeled glycan hydrazide tags.
2) The method involves releasing N-linked glycans from glycoproteins using PNGase F, then derivatizing the glycans from two samples with either a light or heavy tagged reagent.
3) The tagged glycans are mixed, analyzed by LC/MS, and ratios of light and heavy glycan pairs are calculated to quantify differences between the two samples after correcting for isotopic overlap.
This document presents the results of a study aiming to develop selective inhibitors of BACE2 using computer-aided drug design. BACE2 is homologous to BACE1 but is predominantly expressed in peripheral tissues and may serve as an alternative protease in APP processing. The study validated docking programs for reproducing binding poses of BACE1 and BACE2 inhibitors and evaluated scoring functions for virtual screening. A BACE2 homology model produced better virtual screening results than crystal structures. Future work will involve virtual screening larger libraries and designing new scaffolds for selective BACE2 inhibition.
The document discusses protein purification techniques used in research and industry. It describes a protein expression and purification series that teaches students to express and purify the protein dihydrofolate reductase (DHFR) using different modules, including bacterial cell culture, affinity chromatography using nickel beads, and size exclusion chromatography to remove imidazole. The series mimics real-world protein purification workflows and allows students to purify a non-colored protein and analyze it using techniques like SDS-PAGE electrophoresis and enzymatic assays.
Enhanced electrophoretic resolution of monosulfate GAG disacchride isomers on...Yong Zhang
This document describes research on improving the separation of monosulfate glycosaminoglycan disaccharide isomers by microchip electrophoresis. Key findings include:
1) Addition of 1,4-dioxane (DO) to the running buffer dramatically improved resolution of the isomers, likely due to solvation effects.
2) Methylcellulose was used to suppress electroosmotic flow and analyte adsorption to the poly(methyl methacrylate) microchip surface.
3) Optimization of buffer pH, addition of beta-cyclodextrin, and concentration of 1,4-dioxane enhanced resolution of the monosulfate isomers.
4) Under optimized conditions,
This document summarizes research into the HemQ enzyme from Staphylococcus aureus, which catalyzes the final step in the biosynthesis of heme b in some bacteria. The researchers sought to identify reaction intermediates on the pathway from coproheme III to heme b. They found that the first decarboxylation rapidly produces harderoheme III as an intermediate, while the second decarboxylation controls the overall rate. Both harderoheme isomers III and IV reacted when bound to HemQ. While H2O2 is the presumed biological oxidant, peracetic acid could also drive the reaction, suggesting possible iron-mediated reaction mechanisms.
1) Rv3717 is a novel N-acetylmuramoyl-L-alanine amidase enzyme from Mycobacterium tuberculosis that was determined to have a single-domain structure at 1.7 Angstrom resolution.
2) Unlike other bacterial autolysins, Rv3717 lacks a separate cell-wall binding domain and instead uses its net positive charge for substrate binding.
3) The crystal structure revealed a flexible hairpin turn that partially occludes the active site, which may be involved in autoregulation of enzymatic activity similar to other bacterial amidases.
2D gel electrophoresis separates proteins based on their isoelectric point (pI) in the first dimension and molecular weight in the second dimension. Amino acids can exist as zwitterions and act as both acids and bases, donating or accepting protons. The isoelectric point is calculated using the Henderson-Hasselbalch equation and titration curves, taking into account the pKa values of amino acid side chains. Detection methods for proteins on 2D gels include staining with dyes or radioactive labeling.
Chromium-induced growth inhibition and alteration of biochemical parameters i...ijtsrd
The hydroponically grown plants of Ocimum basilicum L. were exposed to varying levels of K2Cr2O7 (0, 5, 10, 25 -µM). The plants were tested for various morphological and biochemical parameters on 3rd and 5th day after treatment. Chromium (Cr) resulted in reduction of plant length and biomass. The deleterious effects of the hexavalent chromium on O. basilicum were further confirmed by the reductions in chlorophyll a and b contents, soluble protein and while as the free amino acid and proline contents were increased. The study concludes that chromium causes stress in the Ocimum basilicum plants and thus alters various morphological and biochemical parameters. Ruqaya Jabeen"Chromium-induced growth inhibition and alteration of biochemical parameters in Ocimum basilicum L." Published in International Journal of Trend in Scientific Research and Development (ijtsrd), ISSN: 2456-6470, Volume-2 | Issue-2 , February 2018, URL: http://www.ijtsrd.com/papers/ijtsrd8329.pdf http://www.ijtsrd.com/chemistry/other/8329/chromium-induced-growth-inhibition-and-alteration-of--biochemical-parameters-in-ocimum-basilicum-l/ruqaya-jabeen
This document reports on the synthesis and characterization of polyethyleneimine-anchored copper(II) complexes and their in vitro DNA binding studies and cytotoxicity. Specifically, it synthesized copper(II) complexes containing 1,10-phenanthroline and L-tyrosine ligands bound to a branched polyethyleneimine polymer. It characterized the complexes using various techniques and studied their binding to calf thymus DNA. It found that the complex with the highest degree of copper(II) coordination bound most strongly to DNA. Finally, it evaluated the cytotoxic activity of this complex against MCF-7 breast cancer cells.
This document describes a new capillary zone electrophoresis (CZE) method for the simultaneous quantitative determination of β-N-oxalyl-L-α,β-diaminopropionic acid (β-ODAP) and homoarginine in Lathyrus sativus (grass pea). The method uses a new sodium borate-sodium sulfate run buffer at pH 9.2. It was found that the ratio of α- and β-ODAP isomers changes from 34.5/65.5 to 28.6/71.4 as the pH increases from 3.0 to 11.0. The method allows for the fast, simple, and sensitive determination of β-OD
This study assessed the neurotoxicity of cobalt (Co) on SH-SY5Y human neuroblastoma cells in vitro. The results showed that CoCl2 reduced cell viability in a dose-dependent manner when measured by MTT and NR assays at 24, 48, and 72 hours. CoCl2 treatment caused cell shrinkage and apoptosis. Antioxidants provided some protection against Co toxicity at lower concentrations but not at higher concentrations, suggesting oxidative stress may not be the main mechanism of toxicity. Overall, the results indicate that Co is toxic to neurons and this toxicity could relate to neurological symptoms reported in patients with cobalt-chromium hip implants who have elevated blood Co levels.
Nucleoside analogues synthesis using natural phosphate doped with i2 (npi2) i...Alexander Decker
This document summarizes research on the synthesis of nucleoside analogues using natural phosphate doped with iodine (NP/I2) as a catalyst. Several D-ribonucleosides were prepared from 1-O-acetyl-2,3,5-tri-O-benzoyl-β-D-ribofuranoside and silylated nucleobases under mild conditions using NP/I2. Yields of the desired nucleosides ranged from 35-62% depending on the nucleobase and amount of iodine used. NP/I2 was shown to be a more effective solid catalyst than silica or alumina doped with iodine. The reaction is proposed to
Proteinase K is a serine protease isolated from a fungus that is able to digest keratin. It has broad substrate specificity and can degrade many native proteins even in the presence of detergents. Proteinase K is commonly used in molecular biology to digest unwanted proteins from nucleic acid preparations. It works best at pH 7.5-8.0 and 37°C, and is typically used at concentrations of 50-200 μg/ml for 30 minutes to 18 hours. Proteinase K is stable and active over a broad range of conditions.
This document summarizes the total synthesis of 2,6-dideoxy-2,6-imino-7-O-β-D-glucopyranosyl-D-glycero-L-gulo-heptitol hydrochloride (8), a potent inhibitor of α-glucosidases. The key steps involve homologation and amination of 2,3,4,6-tetra-O-benzyl-D-glucopyranose (1) to form the protected amine 4. An intramolecular cyclization of 4 catalyzed by mercuric acetate formed the piperidine ring. Glycosylation of aglycon 6 with acetob
This document describes a study characterizing a novel gene cluster involved in the degradation of 4-chlorocatechol by Pseudomonas reinekei MT1. The researchers found that during growth on 5-chlorosalicylate, a novel (chloro)catechol 1,2-dioxygenase (C12OccaA) and a novel (chloro)muconate cycloisomerase (MCIccaB) were induced. MCIccaB was found to transform 3-chloromuconate into equal amounts of cis-dienelactone and protoanemonin, acting as a functional intermediate between known chloromuconate cycloisomerases and muconate cycloisomerases
ABSTRACT- L-Ascorbic acid derivatives was synthesized on treatment with acetone and acetyl chloride afforded 5,6-acetal of L-ascorbic acid then benzylation of C-2 and C-3 hydroxyl groups of the lactone ring was accomplished using K2CO3 and benzyl bromide in DMF, then deblocking of the 5,6-O,O-protected derivative of L-Ascorbic acid with acetic acid and methanol gave 2,3-O,O-dibenzyl-L-Ascorbic acid. Subsequently mono-tosylation at 6 position of 2,3-O, O-dibenzyl-L-Ascorbic acid was carried out with addition of p-toluenetosylchloride (PTSC) in Pyridine and MDC solvent medium gave 2,3-O,O-dibenzyl-6-O-tosyl-L-Ascorbic acid. All the structures were characterized by 1H NMR, 13C NMR and Mass Spectroscopy.
Key-words- L-Ascorbic acid, 5,6-Acetal, Benzylation, Hydrolysis, Tosylation
1. Researchers developed a Si-tag fusion protein containing Si-tag, RGD, and His-tag to enable delivery of biomolecules like RGD onto silica surfaces for tissue engineering applications.
2. They expressed the fusion protein in E. coli and tested various purification methods, finding that rapid purification using silica nanoparticles was more effective than silica gel or IMAC.
3. Functionality assays using immunostaining and binding to silica nanoparticles confirmed the fusion protein retained its ability to bind silica surfaces. Further optimization of purification conditions is still needed to improve protein yield.
Expression of Genetically Engineered Chitinase Gene of Pyrococcus furiosusIJERDJOURNAL
ABSTRACT: Wild-type Pyrococcus furiosus is most likely unable to grow on chitin in the natural biotope due to a nucleotide insertion which separates the chitinase gene into two ORFs, whereas a genetically engineered strain with the deleted nucleotide is able to grow on chitin. In the latest studies, the recombinant enzyme activity against the crystal chitins was examined. But there are still some conflictions. In our study, to shed a light on whether the construct composed of a catalytic domain and a chitin binding domain show any activity against crystalline chitin, the construct was created in the pET 28b (+) expression vector and expressed in Escherichia coli. The chitinase with an approximately 55 kDa molecular weight was determined. The activity of the enzyme was measured spectrophotometrically. Despite the presence of enzyme activity against the colloidal chitin, no significant activity against the crystal chitin has been measured.
This document summarizes a study that evaluated a glycopeptide esterification method to better estimate sialylation levels and differentiate sialic acid linkages in monoclonal antibodies by MALDI-TOF mass spectrometry. Glycopeptides isolated from tryptic digests of monoclonal antibodies were ethyl esterified. Esterification neutralized sialic acids and decreased metastable fragmentation, allowing more accurate quantification of sialylation. Esterification patterns also enabled differentiation of α2,3 and α2,6 sialic acid linkages. The method was demonstrated to be fast, simple, and effective for monoclonal antibody glycopeptide analysis.
The document summarizes research characterizing the radical S-adenosyl-L-methionine (SAM) epimerase NeoN. NeoN catalyzes the specific epimerization of carbon C-5''' in neomycin C to produce neomycin B. The goal is to determine NeoN's structure using X-ray crystallography to understand its catalytic activity. Initial experiments cloning the neoN gene from Streptomyces fradiae and expressing the protein are presented, though yields were low. Future work will optimize cloning and crystallization to elucidate NeoN's structure and characterize residues involved in substrate recognition.
Simple methods for forming protein monolayers lab on-a-chip 2013AnteoDx
1. Mix&Go is a novel surface chemistry that uses metal polymers to activate surfaces for protein binding. It forms thin films below 5 nm that allow proteins to bind as mono-layers while maintaining stability and function.
2. Experiments showed that Mix&Go could activate a variety of surfaces for protein binding, including glass, polystyrene, and gold. It formed stable mono-layers of proteins like streptavidin and antibodies.
3. Mix&Go performed better than conventional methods in assays, requiring less capture protein to achieve similar signals. This suggests it forms cleaner mono-layers versus stacked multi-layers with other methods. Mix&Go could enable new biosensor designs requiring stringent protein-surface interfaces.
- The document examines the role of membrane potential in the biogenesis of cytochrome c oxidase subunit II, a mitochondrial gene product.
- Using an in vitro mitochondrial translation system, the authors find that accumulation of unprocessed subunit II precursor (pre-II) occurs when mitochondrial gene products are synthesized under conditions that prevent formation of a normal membrane potential.
- The majority of pre-II generated in this way is inserted into the lipid bilayer, as judged by resistance to sodium carbonate extraction, indicating that membrane potential is required for a step in subunit II biogenesis other than precursor insertion into the membrane.
Dynamic modification of PMMA chips using PVA for GAG disaccharide isomer sepa...Yong Zhang
This document describes a microchip electrophoresis (MCE) method for separating unsaturated disaccharides from glycosaminoglycans (GAGs) using poly(methyl methacrylate) (PMMA) microchips dynamically coated with poly(vinyl alcohol) (PVA). PVA coating was shown to increase the hydrophilicity of the PMMA surface and reduce nonspecific adsorption. Using PVA-coated PMMA chips, two pairs of GAG disaccharide isomers (nDi-diSB/nDi-diSD and nDi-0S/nDi-HA) were baseline separated within 130 seconds by MCE for the first time. The dynamic PVA coating approach improves MCE resolution for
Interplay between Metabolism and EpigeneticsAbhishek M
Metabolism influences epigenetics through the production of intermediary metabolites that serve as substrates or cofactors for epigenetic enzymes. Key metabolites such as acetyl-CoA, S-adenosyl methionine (SAM), alpha-ketoglutarate (α-KG), nicotinamide adenine dinucleotide (NAD+), and flavin adenine dinucleotide (FAD+) fuel the activities of histone modifying enzymes and DNA methyltransferases. Changes in metabolite levels can therefore alter the epigenetic landscape by impacting chromatin structure and gene expression. Nutrients also influence this interplay, as they directly impact metabolism and the availability of metabolites that regulate the ep
Interplay between Metabolism and EpigeneticsStudy Buddy
This ppt will tell you about the relationship between metabolism and epigenetics. How diiferent metabolites affects epigenetics, how nutrition plays a role in it, etc.
GRAS proteins expression and purification Mesele Tilahun
The document summarizes the production and analysis of the GRAS protein Os-SCL7. It describes how the gene encoding the GRAS domain of Os-SCL7 was cloned and expressed in E. coli. The protein was then purified using nickel affinity chromatography and size exclusion chromatography. Sequence analysis revealed the protein is 378 amino acids with a predicted molecular weight of 41.5 kDa. Potential cleavage sites for specific proteases were also identified from the amino acid sequence.
The document describes the process of purifying the elongation factor LepA/EF4 protein from E. coli. The gene for EF4 was transformed into E. coli cells using a plasmid. The cells were then lysed using sonication and the EF4 protein was purified from the cell lysate using affinity chromatography and its hexahistidine tag. The concentration of the purified EF4 protein was determined to be 0.57 μg/μL using a Bradford assay and its molecular weight was found to be ~69 kDa by SDS-PAGE. Secondary and tertiary structural analysis using circular dichroism and fluorescence spectroscopy yielded thermodynamic values for EF4 protein denaturation.
This document summarizes research investigating the biosynthesis and processing of succinate dehydrogenase (SDH) subunits in cultured mammalian cells. The key points are:
1. Antisera were produced against purified bovine heart SDH and its large and small subunits, which detected precursor and mature forms of the subunits in rat, pig, and bovine cell lines.
2. In pig kidney cells, newly synthesized precursors of the large and small SDH subunits were detected that were 1000-2000 and 4000-5000 Da larger than the mature forms, respectively.
3. Pulse-chase experiments showed the precursor forms were fully processed to the mature subunits within 45 minutes when uncouplers of
This document discusses materials and methods used in a study involving the chemical fipronil and zinc. Twenty male albino rats were divided into four groups of five rats each: a control group, a zinc group that received zinc supplementation, a fipronil group exposed to the insecticide fipronil, and a combination group exposed to both zinc and fipronil. Biochemical assays were conducted to assess oxidative stress markers like superoxide dismutase, catalase, glutathione peroxidase, glutathione-S-transferase, glutathione, lipid peroxidation, and total protein in the rats. Chemicals used including fipronil and zinc sulfate were obtained from reputable suppliers. Kits for the biochemical assays were purchased from a diagnostic
- The researchers created homology models of the Mitotic Checkpoint Complex (MCC) proteins Cdc20p, Mad2p, and Mad3p from budding yeast using an existing crystal structure from fission yeast as a template.
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PROMOTION AND SUPPRESSION OF THERMAL AGGREGATION OFβ-LACTOGLOBULIN BY ARGININ...cscpconf
Bovine β-lactoglobulin (β-lg), consisting of pronounced β-sheet content, have been chosen as a model protein which on prolonged thermal treatment forms large molecular aggregates similar
to Alzheimer’s type amyloid fibrils. The effects of L-arginine (free base) in thermal aggregation
process of β-lg were monitored at varying concentrations. Concentration dependent opposite
behaviour has been reported here for the first time where 0.2-0.3 M concentration was optimized as an apparent critical concentration above which arginine acts as a suppressor and
at below it behaves as a promoter of aggregation of β-lg. olubility study and SDS-PAGE pattern followed by densitometric analysis shows this fact. Solution behaviour of arginine and
its self assemblyformation were evidenced with the help of circular dichroism (CD) studies. The delocalized pi-pi (п→п) type of interaction is proposed to foster the energy stabilization during the attainment of planarity of the molecules accompanied with the self-clustering of arginine molecules.
Gene synthesis involves chemically synthesizing DNA without a template by adding nucleotides one by one. It is the basis of synthetic biology. The key steps in gene synthesis are sequence design, oligo synthesis, gene assembly, sequence verification, and preparing the synthetic DNA. Errors can be introduced at each step, so sequences must be verified before use. Gene synthesis is used widely in research to study biological functions and develop applications like DNA vaccines.
The arrangement of the large (70,000 Mr) and small (30,000 Mr) subunits of succinate dehydrogenase (SDH) in the mitochondrial inner membrane was investigated using limited proteolysis and immunoblotting. Both subunits were resistant to proteinase treatment when the inner membrane integrity was preserved, suggesting neither subunit is exposed at the cytoplasmic surface. The small subunit appears to protrude into the matrix compartment, as it is extensively degraded but no membrane-associated fragment is observed. The large subunit interacts with the matrix side via two distinct domains that are detected as stable membrane-associated fragments after proteinase treatment, though one domain can be further degraded. This suggests the large subunit membrane interaction occurs via two regions,
1. The structure of the scaffold for
cellular growth in-vitro is imperative
for proper cellular adhesion and
proliferation. Previous studies
introduced an electrospun,
mesoporous silica nanofiber (SNF) as
potential scaffold.1 The biodegrability
and biocompatibility of SNF makes it
an optimal scaffold for recombinant
fusion protein Sitag/RGD/His-tag.
Fusion Protein
Taniguchi et al discovered an E. coli
ribosomal L2 protein which has
strong affinity for silica, termed silica
binding protein (Si-tag).2 Attaching
Si-tag to a cell attachment domain
(RGD) as well as a purification
domain (His-tag) allows for the
building of fusion protein for the use
of tissue engineering on SNF.
Scaffold
Lysing is the process of breaking open a cell to extract what is needed on the
inside. There are many different methods to lysing cells; the main categories
include: mechanical, high frequency sound waves, freeze/thaw, chemical, and
liquid homogenization (Fig. 3). Different methods and lysing times were tested to
find the optimal condition to break open the cell and extract the most protein into
the supernatant.The methods tested for lysis include freeze thaw/B-per (Fig. 4), B-
per /lysozyme (Fig. 5), and TE (Tris and EDTA buffer )/lysozyme (Fig. 6). B-Per is a
detergent in a tris buffer. Detergent lysis solutions work by breaking open the lipid
barrier surrounding cells and disrupting interactions and bonds within the cell.
Figure 14. PC12 Cells Differentiating on Cover Slip, PLL, and SB Protein. Cells were exposed to
differentiation media and coated on three surfaces (A) Image of PC12 cells coated on plain
coverslip as a negative control; (B) cells coated on Poly-L-Lysine (PLL) as a positive control; (C)
2DIV and (D) 6DIV PC12 cells coated on Si-RGD-His tag fusion protein.
Cell/ PC12
Purification and Lysis of the Functional Si/RGD/His-tag Fusion Protein
Adam Hildebrandt, Anna Augustine, Samantha Brennan
Supervisor Dr. Mary Ann Yang
His-tag Purification
References
1. Chen WS, Hsieh PH, Yang WN, et al. Chemically modified electrospun silica
nanofibers for promoting growth and differentiation of neural stem cells. J Mater
Chem B. 2014;2:1205–1215.
2. Taniguchi K. 2006. The Si-Tag for Immobilizing Proteins on a Silica Surface. 96. 1023-
1029.
3. Polyhistidine-tag. (n.d.). Retrieved April 25, 2016, from
https://en.wikipedia.org/wiki/Polyhistidine-tag
4. Ikeda T., Ninomiya K., Hirota R., Kuroda A. 2010. Single-step affinity purification of
recombinant proteins using the silica-binding Si-tag as a fusion protein. 71. 91-95.
5. https://www.thermofisher.com/us/en/home/life-science/protein-biology/protein-
biology-learning-center/protein-biology-resource-library/pierce-protein-
methods/traditional-methods-cell-lysis.html
6. Coyle B., Baneyx F. 2014. A Cleavable Silica-Binding Affinity Tag for Rapid and
Inexpensive Protein Purification. Biotechnology and Bioengineering. 111:10.
7. Ikeda T et al. The silica-binding Si-tag functions as an affinity tag even under
denaturing conditions. 2010. Protein Expression and Purificiation. 77: 173-177.
Lysis
A B
C D
Once lysing and purification is optimalized, the protein can be coated onto
glass coverslips. The protein then will allow PC12 cells to sit down on the
scaffold. Which in turn will allow the PC12 cells to grow and differentiated on
the coverslip.
Fig. 4. Purifcation of Sitag/RGD/His-tag fusion protein
utilizing His-tag’s affinity for Nickel.
Fig. 1. Construct of SNF, demonstrating
the three dimensional configuration
Fig 2. Construct of the Sitag/RGD/His-tag
fusion protein
Project Goals
This poster focuses on the experimental lysis and purification techniques that
were done to obtain the Sitag/RGD/His-tag fusion protein. Purified fusion
protein can then be used as a functional portion of the tissue engineering
scaffold.
Affinity tags, generally fused to the N- or C- terminus of expression targets are used to purify desired proteins.6 A
polyhistidine-tag (Histag) is an amino acid motif that consists of at least six histidine (His) residues and is used to
purify recombinant proteins expressed in E. coli.3 In combination with an IMAC column and an affinity resin, the
recombinant Sitag/RGD/His-tag fusion protein is able to be purified (Fig. 4). The resin contains high affinity
transition metal ions immobilized on a solid support through a coordinating ligand.6 Soluble cellular lysates are
added to the column, centrifuged, washed with a salt buffer, and eluted. Washing should remove all non-specific
cellular proteins. Elution, using imidazole, which is a high salt, elutes the protein by competing with binding spots
on the nickel resin. Subsequent to elutions, purified Sitag/RGD/His-tag fusion protein should be obtained.
In context of tissue engineering, it is imperative the Sitag/RGD/His-tag fusion protein is pure.
Any residual bacterial proteins deems the samples unusable for potential neural growth for
patients. Figure 5 see to the left shows the idea schematic for protein purification. Elution lanes
show no presence of residual protein. Figure 6 shows the initial results of purification. The
presence of residual proteins in the elution lanes led us to increase the number of washes, in
hopes to wash away more non-specific proteins. Figure 7 shows the results after increasing the
number of washes from 3 to 6. The presence of proteins still in the elution lanes even after the
increase in washes led to the change in induction concentration from 1.0 mM IPTG to 0.5 mM.
The thought was the higher IPTG concentration was causing protein to coagulate and therefore
caused the Sitag/RGD/His-tag fusion protein to be unable to bind with the nickel resin. The
result of poor yield, residual proteins, and expensive equipment led to moving to a different
purification method.
Fig. 7. Purification with increased washes to
six to rid non-specific proteins
Fig. 5. Ideal schematic of His-tag purification Fig. 6. SDS-PAGE gel of His-tag purification.
Fig. 8. His-tag purification using decreased IPTG
concentration.
Sitag Purification
The strong bindiing of silica is attributed to the unusual basicity, positive residues and disorderd structure of the Sitag
polypeptide.6 Sitag, an intrinsically disordered (ID) protein has fluctuating structures which allows for flexibility and optimal
binding to increase its intermolecular forces. Its positively charged side chain and residues (lysine) allow it to bind to the negatively
charged silica surfaces. Sitag also binds through its apolar side chains and the hydrophobic silica surface. Previous studies showed
that even under denaturing conditions (urea), Sitag remains bound, confirming that the binding is independent of structure.7 This
purification method utilizes silica nanoparticles and the stated Sitags affinity to them. Varying elution methods were tested.
Elution with MgCl2 Elution with L-lysine
Previous studies done by Ikeda showed that MgCl2 is able to elute
the fusion protein by interfering with Sitag’s binding to silica (Fig
9).4 It was found that divalent cations such as Mg2+ and Ca2+ could
release Sitag at a high concentration (2M); studies found however
that because Mg2+ has a higher ionic potential, it is more effective
than Ca2+. Due to the little Sitag released when using NaCl, it
suggests that divalent cations play an important role in
dissociating the protein.
Fig. 12. A) Shows the washes and elution flow-
throughs from purification while B) shows
concentrate and SNPs. The fusion protein was
unable to be dissociated from the SNPs. These
figures show the effects of different
concentrations of L-Lysine on dissociating the
Si-Tag fusion protein from silica nanoparticles.
Fig. 13. Increased washes and incubation time.
Fusion protein was able to be eluted form SNPs
via L-lysine.
Fig 10. There is high
levels of gel
disruption in this
figure due to high
amounts of salt
remaining in the
samples. It is
hypothesized that the
high salt
concentration
wouldn’t be optimal
for neuron
differentiation.
Future Work
Fig 9.
Results from
Ikeda
studies. Lane
1: control
Lane 5M
NaCl Lane 3:
2 M MgCl2
Lane 4: 2 M
CaCl2, Lane
5: 1 N HCl
and Lane 6:
1N NaOh
Divalent cations such as MgCl2 have a much higher ionic potential
than Na+ ions; therefore divalent cations are electrostatically
attracted to negatively charged silica surfaces more strongly than
monovalent Na+. This suggests that Sitag proteins are
electrostatically absorbed on silica surfaces and are then
dissociated by an ion exchange effect where Mg2+ ions act as
competition for the silanoal groups. 4 This competition allows for
the dissociation of the fusion protein.
Figure 3. Description of Lysing Methods. A table
explaining how different techniques of lysing work and
what is needed for each process.
Figure 4. SDS-PAGE Results from Lysing E.coli Cells using B-
Per/Lysozyme 1mg/mL and Dry Ice Freeze Thaw. SDS-PAGE
comparing lysing with dry ice and without. Different lysing times
of 30 and 60 minutes were also tested. All samples were lysed
with B-per/lysozyme 1mg/mL as well.
Figure 5. SDS-PAGE Showing Protein Sample Results
from Testing Different Lysing Conditions. Two different
conditions of TE/lysozyme buffer with final concentration
of 1mg/mL and B-per/DNase/ lysozyme buffer with a
1mg/mL final concentration were tested for optimal lysing
on Rosetta E.coli. All conditions were incubated at 37˚C
water. bath for either 30 or 60 minutes
Figure 6. SDS-PAGE Results From TE/Lysozyme V.s B-
Per/Lysozyme at Different Concentrations. Optimal cell lysis was
tested by comparing the effectiveness of increasing B-
Per/Lysozyme concentration from 200mg/mL to 1.0mg/mL.
These conditions were tested against TE/Lysozyme at 1.0mg/mL
final concentration. . All conditions were incubated at 37˚C water.
bath for 30 minutes.
If further testing isn’t indicative of Si-Tag purification; this
research might shift towards using a silica gel instead of
using silica nanoparticles. This method is proven to be
quickly purify the Car9 fusion protein.6 It is hypothesized
that the nanoparticles might instigate too strong of an
association for effective elution using L-Lysine. Silica gel is
what is used in.6 An apparatus will be assembled with two
plastic syringes with a valve connecting them (figure 15).
Silica gel is placed on top of glass wool and then is
immersed in wash buffer.6 Next, the cellular lysate can be
added to the system. As the suction pressure is being
applied, the fusion protein should theoretically bind to the
silica while all other unwanted items can flow through the
system by simply pulling the bottom syringe. To elute the
fusion protein from the apparatus, L-Lysine will be used to
elute the fusion protein to effectively purify it. The suction
pressure applied from the syringe at the bottom effectively
increases flow rate as well as purification time.6
Figure 15. Two syringe
system adapted from
(Coyle et al., 2014). This
system uses silica gel and
pressures from the syringe
to purify fusion proteins.
The first purification ran using L-lysine (Fig 10) shows that L-
lysine was unable to dissociate the protein. In efforts to find a
cheap and effective method for eluting the fusion protein from
silica, it was successfully proven that l-lysine effectively
dissociates GFPmut2-Car9 from silica.6 It is suggested in this
article, that the binding of the Car9 fusion protein involves
electrostatic interactions. Electrostatic interactions are
molecular associations that occur between basic and acidic
regions of molecules due to positive and negative electron
charges. In solution, the silica surface is converted in to silanol
groups; it is these silanol groups that interact with the basic
regions of the desired fusion protein.6 In addition to
electrostatic interactions, the association of the fusion protein
to silica also contains hydrophobic organization between the
central phenylalanine and siloxane groups.6 L-Lysine is a
positively charged amino acid that can compete for the silanol
binding sites through electrostatic interactions. Also, L-Lysine
contains an R group that is hydrophobic in nature. In tandem,
these properties theoretically allow for L-Lysine work to act as
an eluent for the Si-Tag fusion protein. It was discovered that a
1M solution of L-Lysine effectively eluted the Mut2-Car9 fusion
protein from silica with a purity of about 80%.6 The rational
behind testing L-Lysine elution is that it is a cheap, effective
and fast method for eluting fusion proteins.6 Coyle states that
using L-Lysine for elution costs two dollars per run in worst-
case scenarios. This is around ten times cheaper than a
previously used His-Tag purification. Also, purification of the
fusion protein could be obtained from cellular lysate in as little
as 15 minutes. The next logical step was to elute the Si-Tag
fusion protein from the silica nanoparticles using 1M L-Lysine.
Early results indicated that the interactions between the silica
nanoparticles and the Si-Tag fusion protein were too strong for
elution with 1M L-Lysine. Increased concentrations of L-Lysine
indicated some purification. Although some bacterial proteins
still remained.
Results indicated that B-PER acts as the best detergent (Fig. 5) when referencing
the bands for the supernatant. Freeze-thaw cycles showed no improvement for
lysis and was therefore not used in future experiments (Fig. 4). Increasing B-PER
concentration to 200 mg/ml showed no improvement in the recovered
supernatant fusion protein.
Figure 11. Adapted from (Coyle et al.,
2014) Si-Tag fusion protein association
with silanol groups located on the silica
surface
A
B