Project done as a final presentation for Experimental Biochemistry. The project was designed and proposed by me and performed by myself and Lauren Pioppo.
The IOSR Journal of Pharmacy (IOSRPHR) is an open access online & offline peer reviewed international journal, which publishes innovative research papers, reviews, mini-reviews, short communications and notes dealing with Pharmaceutical Sciences( Pharmaceutical Technology, Pharmaceutics, Biopharmaceutics, Pharmacokinetics, Pharmaceutical/Medicinal Chemistry, Computational Chemistry and Molecular Drug Design, Pharmacognosy & Phytochemistry, Pharmacology, Pharmaceutical Analysis, Pharmacy Practice, Clinical and Hospital Pharmacy, Cell Biology, Genomics and Proteomics, Pharmacogenomics, Bioinformatics and Biotechnology of Pharmaceutical Interest........more details on Aim & Scope).
Effect of estradiol -17 β on arachidonic acid metabolism in sheep uterus: in ...iosrjce
The effect of estradiol-17 β on Arachidonic acid (AA) metabolism in non-pregnant sheep uterus was
studied under in vitro conditions. On incubation of uterine slices with estradiol-17β, the levels of prostaglandins
were altered but not Lipoxygenase (LOX) products. Based on their analysis on conventional TLC technique, the
Cyclooxygenase (COX) products PGF2α, 6-keto PGF1α and PGE2 were shown to be altered over an incubation
period of 0 to 120 minutes. The LOX products, HPETEs and HETEs did not show any change upon incubation
with estradiol-17β. This study gives a preliminary understanding of role of estradiol on AA metabolism.
The IOSR Journal of Pharmacy (IOSRPHR) is an open access online & offline peer reviewed international journal, which publishes innovative research papers, reviews, mini-reviews, short communications and notes dealing with Pharmaceutical Sciences( Pharmaceutical Technology, Pharmaceutics, Biopharmaceutics, Pharmacokinetics, Pharmaceutical/Medicinal Chemistry, Computational Chemistry and Molecular Drug Design, Pharmacognosy & Phytochemistry, Pharmacology, Pharmaceutical Analysis, Pharmacy Practice, Clinical and Hospital Pharmacy, Cell Biology, Genomics and Proteomics, Pharmacogenomics, Bioinformatics and Biotechnology of Pharmaceutical Interest........more details on Aim & Scope).
Effect of estradiol -17 β on arachidonic acid metabolism in sheep uterus: in ...iosrjce
The effect of estradiol-17 β on Arachidonic acid (AA) metabolism in non-pregnant sheep uterus was
studied under in vitro conditions. On incubation of uterine slices with estradiol-17β, the levels of prostaglandins
were altered but not Lipoxygenase (LOX) products. Based on their analysis on conventional TLC technique, the
Cyclooxygenase (COX) products PGF2α, 6-keto PGF1α and PGE2 were shown to be altered over an incubation
period of 0 to 120 minutes. The LOX products, HPETEs and HETEs did not show any change upon incubation
with estradiol-17β. This study gives a preliminary understanding of role of estradiol on AA metabolism.
Expression, purification and spectroscopic characterization of the cytochrome...John Clarkson
K.J. McLean, M.R. Cheesman, S.L. Rivers, A. Richmond, D. Leys, S.K. Chapman, G.A. Reid, N.C. Price, S.M. Kelly, J. Clarkson, W.E Smith & A.W. Munro, “Expression, Purification and Spectroscopic Characterization of the Cytochrome P450 CYP121 from Mycobacterium Tuberculosis”, J. Inorganic Biochemistry, 91, 527-541, 2002.
(48)Screening and identifiation of Bacillus sp. isolated from traditional Vie...minhdaovan
Fibrinolytic enzymes produced by microorganisms have been attractive in prevention and
treatment of cardiovascular diseases (CVDs) by their low-cost and safety. This study focused
on screening for the existence of firinolytic enzymes in Vietnamese traditional fermented
soybean paste products and isolation and identifiation of related bacteria. Sixteen fermented
soybean paste samples were collected over three regions of Vietnam in which seven samples
gave the positive results on firinolytic enzyme activity. Miso (MS) and Green Chili (GC)
samples had the highest firinolytic enzyme activities (1.81 and 0.77 FU/g, respectively).
According to morphological features, four strains of bacteria were isolated and all of them
were found to produce firinolytic enzymes. The enzyme activities produced by four isolated
strains were in a range of 29.7 - 77.9 FU/g after culturing on solid state media for 24 h. The
isolated strains were identifid as Bacillus amyloliquefaciens using 16 rRNA sequence and
phylogenetic analysis with 99% similarity
Immobilization of two endoglucanases from different sourcesIJEAB
Cellulases are a important family of hydrolytic enzymes which catalyze the bond of cellulose and other related cello-oligosaccharide derivates. Industrial applications require enzymes highly stable and economically viable in terms of reusability. These costs can be reduced by immobilizing the cellulases, offering a potential solution through enzyme recycling and easy recovery. The covalent immobilization of enzymes is reported here: one is commercial cellulase from Aspergillus niger and other one is recombinant enzyme, named CelStrep it because was isolated from a new cellulolytic strain, Streptomyces sp. G12,. The optimal pH for binding is 4.6 for both cellulases and the optimal enzyme concentrations are 1 mg/mL and 5 mg/mL respectively. The support for immobilization is a poliacrylic matrix. Experiments carried out in this work show positive results of enzyme immobilization in terms of efficiency and stability and confirm the economic and biotechnical advantages of enzyme immobilization for a wide range of industrial applications.
International Journal of Pharmaceutical Science Invention (IJPSI)inventionjournals
International Journal of Pharmaceutical Science Invention (IJPSI) is an international journal intended for professionals and researchers in all fields of Pahrmaceutical Science. IJPSI publishes research articles and reviews within the whole field Pharmacy and Pharmaceutical Science, new teaching methods, assessment, validation and the impact of new technologies and it will continue to provide information on the latest trends and developments in this ever-expanding subject. The publications of papers are selected through double peer reviewed to ensure originality, relevance, and readability. The articles published in our journal can be accessed online
Advantages of microbial biotransformation of bioactive compounds & microbial ...Radwa Ahmed
advantages of the use of microbial biotransformation in the field of natural products.
The microbial models for mammalian drug metabolism and applications in drug studies
Effect of Isolate of Trichoderma Sp. And Incubation Period to Glucose Productioninventy
This study was carried out to determine the effect of isolates of Trichoderma sp. and the incubation period to glucose production. A glucose produced by the three isolates of Trichoderma sp. (TV.0209, TV.0305 and TV.0710) which were obtained from the collection of the Laboratory of Microbiology, Department of Chemical Engineering of The State Polytechnic of Malang. It was determined that the isolates had a significant correlation in producing glucose with incubation period for 10 days. Data were then analyzed using two-way anova with the alpha of 5% and using Microsoft Excel. Isolate TV.0209 was capable to produce the highest average of a total glucose production (3,616 ppm) when compared with TV.0305 (3,253 ppm) and TV.0710 (2,448 ppm). The optimum incubation period of treatment isolate variables was achieved during 6 days.
Extraction of β-galactosidase and β-glucosidase from the seeds of Tamarindus ...Innspub Net
The enzymes β–galactosidase and β–glucosidase were extracted from the tamarind seeds using different buffers at different pH. Highest activity was obtained with 10 mM sodium acetate buffer, pH 5.6 and 10 mM tris buffer, pH 7.4. The effect of NaCl and Triton X–100 at different concentrations on the extraction of the enzymes indicated 10 mM sodium acetate buffer, pH 5.6 containing 1 M NaCl as a better extractant of the enzyme. The enzyme assay was carried out using p–nitrophenyl–β–D–galactoside and p–nitrophenyl–β–D–glucoside as substrates. Highest enzyme activities were observed on 6th and 24th day of germination. The protein content gradually decreased upto 5th day of germination and suddenly increased on 6th day. However, on subsequent days of germination, the protein content greatly decreased upto 11th day. During the latter period of germination (18th day onwards) the content remained almost constant. The kinetic parameters varied for both β–galactosidase and β–glucosidase. The activity of β–galactosidase was show to have an optimal operating condition at pH 5.5 and a temperature of 500C. The thermostability of the enzyme was in the range of 400C – 700C with the pH stability in the range of 5.0 – 7.0. The Km and Vmax values for pNPGal were determined as 66μM and 2.27nmolesmin-1. In contrast the activity of β–glucosidase was shown to have an optimal operating condition at pH 5.0 and a temperature of 300C. The thermostability of the enzyme was in the range of 270C – 350C with the pH stability in the range of 4.0 – 7.0. The Km and Vmax values for pNPGlu were determined as 121μM and 5.26nmolesmin-1. The presented study is a preliminary work carried out for the standardization of protocols. The purification and characterization of β–galactosidase and β–glucosidase is under progress.
Expression, purification and spectroscopic characterization of the cytochrome...John Clarkson
K.J. McLean, M.R. Cheesman, S.L. Rivers, A. Richmond, D. Leys, S.K. Chapman, G.A. Reid, N.C. Price, S.M. Kelly, J. Clarkson, W.E Smith & A.W. Munro, “Expression, Purification and Spectroscopic Characterization of the Cytochrome P450 CYP121 from Mycobacterium Tuberculosis”, J. Inorganic Biochemistry, 91, 527-541, 2002.
(48)Screening and identifiation of Bacillus sp. isolated from traditional Vie...minhdaovan
Fibrinolytic enzymes produced by microorganisms have been attractive in prevention and
treatment of cardiovascular diseases (CVDs) by their low-cost and safety. This study focused
on screening for the existence of firinolytic enzymes in Vietnamese traditional fermented
soybean paste products and isolation and identifiation of related bacteria. Sixteen fermented
soybean paste samples were collected over three regions of Vietnam in which seven samples
gave the positive results on firinolytic enzyme activity. Miso (MS) and Green Chili (GC)
samples had the highest firinolytic enzyme activities (1.81 and 0.77 FU/g, respectively).
According to morphological features, four strains of bacteria were isolated and all of them
were found to produce firinolytic enzymes. The enzyme activities produced by four isolated
strains were in a range of 29.7 - 77.9 FU/g after culturing on solid state media for 24 h. The
isolated strains were identifid as Bacillus amyloliquefaciens using 16 rRNA sequence and
phylogenetic analysis with 99% similarity
Immobilization of two endoglucanases from different sourcesIJEAB
Cellulases are a important family of hydrolytic enzymes which catalyze the bond of cellulose and other related cello-oligosaccharide derivates. Industrial applications require enzymes highly stable and economically viable in terms of reusability. These costs can be reduced by immobilizing the cellulases, offering a potential solution through enzyme recycling and easy recovery. The covalent immobilization of enzymes is reported here: one is commercial cellulase from Aspergillus niger and other one is recombinant enzyme, named CelStrep it because was isolated from a new cellulolytic strain, Streptomyces sp. G12,. The optimal pH for binding is 4.6 for both cellulases and the optimal enzyme concentrations are 1 mg/mL and 5 mg/mL respectively. The support for immobilization is a poliacrylic matrix. Experiments carried out in this work show positive results of enzyme immobilization in terms of efficiency and stability and confirm the economic and biotechnical advantages of enzyme immobilization for a wide range of industrial applications.
International Journal of Pharmaceutical Science Invention (IJPSI)inventionjournals
International Journal of Pharmaceutical Science Invention (IJPSI) is an international journal intended for professionals and researchers in all fields of Pahrmaceutical Science. IJPSI publishes research articles and reviews within the whole field Pharmacy and Pharmaceutical Science, new teaching methods, assessment, validation and the impact of new technologies and it will continue to provide information on the latest trends and developments in this ever-expanding subject. The publications of papers are selected through double peer reviewed to ensure originality, relevance, and readability. The articles published in our journal can be accessed online
Advantages of microbial biotransformation of bioactive compounds & microbial ...Radwa Ahmed
advantages of the use of microbial biotransformation in the field of natural products.
The microbial models for mammalian drug metabolism and applications in drug studies
Effect of Isolate of Trichoderma Sp. And Incubation Period to Glucose Productioninventy
This study was carried out to determine the effect of isolates of Trichoderma sp. and the incubation period to glucose production. A glucose produced by the three isolates of Trichoderma sp. (TV.0209, TV.0305 and TV.0710) which were obtained from the collection of the Laboratory of Microbiology, Department of Chemical Engineering of The State Polytechnic of Malang. It was determined that the isolates had a significant correlation in producing glucose with incubation period for 10 days. Data were then analyzed using two-way anova with the alpha of 5% and using Microsoft Excel. Isolate TV.0209 was capable to produce the highest average of a total glucose production (3,616 ppm) when compared with TV.0305 (3,253 ppm) and TV.0710 (2,448 ppm). The optimum incubation period of treatment isolate variables was achieved during 6 days.
Extraction of β-galactosidase and β-glucosidase from the seeds of Tamarindus ...Innspub Net
The enzymes β–galactosidase and β–glucosidase were extracted from the tamarind seeds using different buffers at different pH. Highest activity was obtained with 10 mM sodium acetate buffer, pH 5.6 and 10 mM tris buffer, pH 7.4. The effect of NaCl and Triton X–100 at different concentrations on the extraction of the enzymes indicated 10 mM sodium acetate buffer, pH 5.6 containing 1 M NaCl as a better extractant of the enzyme. The enzyme assay was carried out using p–nitrophenyl–β–D–galactoside and p–nitrophenyl–β–D–glucoside as substrates. Highest enzyme activities were observed on 6th and 24th day of germination. The protein content gradually decreased upto 5th day of germination and suddenly increased on 6th day. However, on subsequent days of germination, the protein content greatly decreased upto 11th day. During the latter period of germination (18th day onwards) the content remained almost constant. The kinetic parameters varied for both β–galactosidase and β–glucosidase. The activity of β–galactosidase was show to have an optimal operating condition at pH 5.5 and a temperature of 500C. The thermostability of the enzyme was in the range of 400C – 700C with the pH stability in the range of 5.0 – 7.0. The Km and Vmax values for pNPGal were determined as 66μM and 2.27nmolesmin-1. In contrast the activity of β–glucosidase was shown to have an optimal operating condition at pH 5.0 and a temperature of 300C. The thermostability of the enzyme was in the range of 270C – 350C with the pH stability in the range of 4.0 – 7.0. The Km and Vmax values for pNPGlu were determined as 121μM and 5.26nmolesmin-1. The presented study is a preliminary work carried out for the standardization of protocols. The purification and characterization of β–galactosidase and β–glucosidase is under progress.
Genome-scale in silico atpE gene knockout in Escherichia coli could drive nov...Khadem2016
One of the applications of E. coli genome-scale model is in the biological discovery of underground metabolic functions of partially characterized genes and/or enzymes. Here we report for the first time, a failed prediction of atpE gene knockout of no growth in the most recent E. coli reconstruction iJ01366 model, and a positive experimental growth on glucose, enabling a model-driven biological discovery of the underground metabolic function of this gene in E. coli metabolism. These findings unfolded what could be described as either scope gaps in the reconstruction or true biological gaps (knowledge gaps) on the missing atpE gene function in E. coli metabolism. This study informs other studies that the gaps could be pursued into the E. coli metabolism, leading to a model-driven discovery in the future.
One of the applications of E. coli genome-scale model is in the biological discovery of underground metabolic functions of partially characterized genes and/or enzymes. Here we report for the first time, a failed prediction of atpE gene knockout of no growth in the most recent E. coli reconstruction iJ01366 model, and a positive experimental growth on glucose, enabling a model-driven biological discovery of the underground metabolic function of this gene in E. coli metabolism. These findings unfolded what could be described as either scope gaps in the reconstruction or true biological gaps (knowledge gaps) on the missing atpE gene function in E. coli metabolism. This study informs other studies that the gaps could be pursued into the E. coli metabolism, leading to a model-driven discovery in the future. This can be achieved by using gap filling algorithms (such as GrowMatch, SMILEY
Genome-scale in silico atpE gene knockout in Escherichia coli could drive nov...Khadem2016
One of the applications of E. coli genome-scale model is in the biological discovery of underground metabolic functions of partially characterized genes and/or enzymes. Here we report for the first time, a failed prediction of atpE gene knockout of no growth in the most recent E. coli reconstruction iJ01366 model, and a positive experimental growth on glucose, enabling a model-driven biological discovery of the underground metabolic function of this gene in E. coli metabolism. These findings unfolded what could be described as either scope gaps in the reconstruction or true biological gaps (knowledge gaps) on the missing atpE gene function in E. coli metabolism. This study informs other studies that the gaps could be pursued into the E. coli metabolism, leading to a model-driven discovery in the future. This can be achieved by using gap filling algorithms (such as GrowMatch, SMILEY
In vitro experiments of prokaryotic and eukaryotic antimicrobial peptide cyto...AI Publications
These proteinaceous molecules, called antimicrobial peptides (AMPs), are a varied collection of antimicrobial peptides. The ability of AMPs to combat gut infections necessitates further study of the AMP-GI tract interaction. These peptides need to be tested in vitro for cytotoxicity before they may be considered for use in clinical infections. Using the MTT conversion assay, neutral red dye absorption assay, and a comparison to vancomycin, researchers examined the cytotoxicity of gallidermin, nisin A, natural magainin peptides, and melittin in two gastrointestinal cell types (HT29 and Caco-2). Sheep erythrocyte hemolytic activity was also studied, and the influence of AMPs on paracellular permeability was assessed using transepithelial resistance (TEER) and TEM. Gallidermin, nisin A, magainin I, magainin II, and melittin were the least cytotoxic AMPs. To our knowledge, only Melittin and NIS caused considerable hemolysis. There are two distinct ways that melittin and nisin differ in their ability to kill bacteria. It was the only AMP that had an effect on the permeability of the paracellular space. Intestinal tight junctions and cell–cell adhesion were destroyed by long-term melittin therapy, as were microvilli, cell debris, and cell–cell adhesion. Antimicrobial activity and low cytotoxicity make Gallidermin a promising therapeutic drug. The antibacterial properties of Melittin are limited, but its ability to transport poorly bioavailable medicines may be useful.
Investigating Chemical Chaperones that can improve the stability of Lysozymes...oyepata
Investigating Chemical Chaperones that can improve the stability of Lysozymes
under high thermal temperature.
Sabastine Aliyu Zubairu1, Joseph Oyepata Simeon2, Isaac Ralph Elon1, Mahdi
Mohammed1, Sunday Blessing
A comparative study of procedures for binding of aflatoxin M1 to 1 Lactobacil...Jean Claude Assaf
Several strains of Lactic Acid Bacteria (LAB), frequently used in food fermentation and 20 preservation, have been reported to bind different types of toxins in liquid media. This study 21 was carried out to investigate the effect of different concentrations of Lactobacillus 22 rhamnosus GG (ATCC 53103) to bind aflatoxin M1 (AFM1) in liquid media. AFM1 binding 23 was tested following repetitive washes or filtration procedures in combination with additional 24 treatments as heat, pipetting, and centrifugation. The mixture of L. rhamnosus GG and AFM1 25 was incubated for 18 hours at 37 °C and the unbound AFM1 was quantified by HPLC. The 26 stability of the bacteria-AFM1 complex for both viable and heat treated bacteria was tested. 27 Depending on the bacterial concentration and procedure used, the percentages of bound AFM1 28 by L. rhamnosus GG varied from as low as undetectable to as high as 63%. The highest 29 reduction in the level of unbound AFM1 was recorded for the five washes procedure that 30 involved heat and pipetting treatments. Results also showed that binding was partially 31 reversible and AFM1 was released after repeated washes. These findings highlight the effect 32 of different treatments on the binding of L. rhamnosus GG to AFM1 in liquid matrix.
Cloning, Expression and Purification of Recombinant Interleukin 1 Receptor Antagonist (IL-1RA) in Escherichia coli
http://dx.doi.org/10.21276/SSR-IIJLS.2020.6.3.1
Introduction
Examples of Protein-Ligand interactions
Protein-Ligand Interactions Can Be Described Quantitatively
Cooperative Ligand Binding Can Be Described Quantitatively by Hill Equation
Physical and chemical methods of protein-ligand interaction study
Verification of interactions
Public protein–protein interaction databases
Ligand docking
Conclusions
References
2. Abstract:
Glycine Oxidase, or ThiO, from Bacillus subtilis catalyzes the FAD dependent oxidation of
glycine to imino-glycine in the thiamin biosynthesis pathway. In this pathway, the mechanism in
which iminoglycine and ThiS reaches the active site of ThiG remains unknown (Settembre et al
2003). Elucidation of this step in the pathway can aid in the development of drugs that target
thiamin biosynthesis in several pathogenic prokaryotes, such as P falciparum, the increasingly
resistant protozoan parasite that causes malaria. Here, we use recombinant His-tagged ORF811
from Bacillus muhlenbergius, a close homolog of B. subtilis ThiO, to analyze any protein:protein
interactions among ThiO and other enzymes involved in the passage of iminoglycine to ThiG
during thiamin biosynthesis. A pull-down assay of ORF811 and CFCE of B. muhlenbergius did not
reveal any stable protein:protein interactions with ORF811. Additionally, cross-linking studies
were performed to elucidate any transient interactions; a Western blot of these reactions
indicated possible dimer formation of ORF811, but did not reveal any relevant transient
interactions.
3. Introduction:
Many molecular processes throughout the body, in all tissues, involve the interaction of
multiple proteins. These interactions can range from modification of other proteins, such as
phosphorylation or acylation, to the coupling of mechanistic steps by the close physical contact
of enzymes in a metabolic pathway. The latter can be accomplished by several methods
including the formation of large multi-enzyme complexes, such as pyruvate Dehydrogenase
(McMurry, Begley 2005), or unstable transient protein interactions (Krishnamurthy et al 2011).
Identifying protein-protein interactions has become a new practice of proteomics (Blagoev et al
2003) and can serve to elucidate how complex reactions involving reactive intermediates can
progress at a cellular level.
Recently, a new organism named Bacillus muhlenbergius has been identified as a rare
soil bacterium inhabiting areas in Pennsylvania near Muhlenberg College, Allentown. This
organism is a close relative via genomic sequence of the more ubiquitous B. subtilis. A recent
project has been undertaken by this lab to identify the genomic characteristics of B.
muhlenbergius by elucidating the characteristics of several prokaryotic enzymes in hopes of
identifying enzymes that may be useful for biotechnological advances; one such enzyme is
Orf811. Bioinformatic analysis has identified Orf811 as a close homolog of Glycine Oxidase
(GO)1 from B. subtilis. GO is commonly referenced as ThiO for its oxidative role thiamin
biosynthesis. This enzyme is a homotetrameric protein that catalyzes the FAD dependent
oxidation of glycine to iminoglycine before being shuttled down the anabolic pathway
(Settembre et al 2003). However, several key factors in this progression of the pathway remain
1 3
GO: Glycine Oxidase. BS : Bissulfosuccinimidyl suberate. EDC: 1-ethyl-3-[dimethylaminopropyl]-carbodiimide.
CFCE: Cell-Free Crude Extract. BC: Bait Control. RC: Resin Control
4. unsolved. The imine product of GO is highly reactive in solvent and is readily hydrolyzed (Mortl
2006). This hydrolyzed product is not capable of being used in the pathway as the following
reaction of ThiS requires the imine product to continue. The method by which iminoglycine and
ThiS reach the active site of ThiG remains to be solved.
The solved crystal structure of GO has shown that the enzyme contains a large channel
leading to the active site, which is buried within the hydrophobic core. However, the active site
is solvent exposed, indicating that the imine product cannot leave the active site without being
hydrolyzed (Mörtl et al 2004). Several enzymes that produce reactive intermediates have been
shown to contain structural characteristics that allow transfer of the species to the next active
site without exposure to other cellular components or solvent, such as the indole intermediate
in tryptophan synthase (Raushel et al 2003). This multifunction enzyme performs both portions
of the reaction with a protein ‘tunnel’ throughout the center to shuttle the reactive indole
molecule to the second active site. Other enzymes that produce reactive species have been
found to be part of large, multi-enzyme complexes, allowing the close proximity of the enzymes
in solvent to facilitate the transfer of the reactive species from one active site to another
(McMurry, Begley 2005). Given the reactive nature of iminoglycine and other protein
interactions within the thiamin biosynthesis pathway, such as that between ThiG and ThiS
(Settembre et al 2004), it is likely that some form of intermediate shuttling is occurring in order
to prevent hydrolysis prior to reacting with ThiS.
In addition, the thiamin biosynthesis pathway has long been known as an antibiotic
target for several pathogenic prokaryotes that are unable to salvage vitamin B1, the most
notable being P. falciparum, the infections agent responsible for Malaria (Muller et al 2010).
Malaria was responsible for roughly 781,000 deaths in 2009 alone and is resistant to other
forms of antibiotic treatment as it is an obligate intracellular parasite (WHO 2011). Identifying
possible protein-protein interactions within the thiamin biosynthesis pathway can provide a
novel drug target for antibiotic research against this increasingly resistant pathogen.
In this experiment, we attempted to elucidate the details of this passage of imino-
glycine to ThiS by performing a pull-down assay using His-tagged Orf811 and cell free crude
5. extracts from B. muhlenbergius to identify any stable protein-protein interactions between
other enzymes in the thiamin synthesis pathway. SDS-PAGE analysis revealed no visible
difference from control samples, indicating that no stable interactions had formed. In an
attempt to find any transient interactions between proteins, molecular crosslinkers were used
to identify putative protein-protein interactions involving ThiO. Western blot analysis of
samples crosslinked by BS3 showed possible dimer formation of ThiO, while EDC crosslinking
showed no bands. This study is important in identifying interactions between ThiO and other
enzymes in the thiamin synthesis pathway which can serve to explain how this reaction
progresses beyond the oxidation of glycine without loss of product to hydrolysis. Comparison of
this process in B. muhlenbergius to other prokaryotic organisms can serve to identify
evolutionary characteristics of this new organism and provide drug targets for P. falciparum.
6. Materials and Methods
Chemicals and Equipment
All reaction kits were purchased from Thermo Scientific. Chemicals were purchased from
Qiagen, except for imidazole and Triton X-100 (ACROS), TRIS and 10% Tween 20 (BioRad), and
GelCode Stain and Coomassie Plus Protein Assay Reagent (Thermo Scientific). All high speed
(>10,000g) centrifugation was performed in the Sorvall RC6 High Speed Centrifuge, while slower
speeds were performed in the Eppendorf 5430. Cell Lysis was performed using a BeadBeater
(BioSpec).
Bioinformatics Analysis
A Bioinformatic analysis of Orf811 was performed using ExPASy to obtain a partial
protein sequence from cDNA. The Basic Alignment Search Tool (BLASTP) was used to identify
potential homologs of Orf811 using the translated sequence. GenBank was then used to
identify the genomic context of GO.
Preparation of Bacillus muhlenbergius Cells
Two cultures of B. muhlenbergius cells were grown in 1 liter of minimal media lacking
thiamin (5x M9 salts, Difco casamino acids, 20% glucose) at 30 C for about 48 hours. Cells were
harvested by centrifugation at 10,000xg for 30 min. Cell pellets were lysed by BeadBeating on
ice (12 cycles of beating for 15 seconds, followed by 45 seconds of rest) and centrifuged at
15,000xg to obtain CFCE. The concentration of the crude extract was determined using a
Bradford assay. Bovine serum albumin (BSA) was used for calibration.
Pull-Down Assay:
A pull-down assay of B. muhlenbergius ORF811 was performed using the ProFound
Pull-Down PolyHis Protein:Protein Interaction Kit. Poly-His tagged ORF811 (3.5 mg/ml), a
generous gift from Dr. Keri Colabroy, Muhlenberg College, was used as bait protein, and
prepared cell-free crude extracts of B. muhlenbergius were used as a source of prey proteins.
7. Orf811 and CFCE were combined prior to use in the pull-down to a final protein concentration
of 2.35 mg/ml. This combined protein solution was also used for both cross-linking studies. The
pull-down was performed according to the protocol provided by Thermo Scientific. Briefly,
three 1.0 mL columns were prepared [(Bait Control (+Orf811, -CFCE), Resin Control (-Orf811,
+CFCE), Sample (+Orf811, -CFCE)] and treated with the corresponding proteins. Columns were
incubated at 4°C for 1 hour following the addition of Orf811 and an additional hour after
washing the column using the provided wash solution and treatment with CFCE. All flow
through was collected for analysis. Bound Orf811 was eluted from the columns using Imidazole
Elution Buffer (From kit).
Cross-Linking Studies
Cell-free crude extracts of B. muhlenbergius and purified poly-His tagged ORF811 were
buffer exchanged into 20 mM potassium phosphate monobasic buffer (pH 7.8) using an Econo-
Pac 10 DG gel filtration column. This mixture was cross-linked with bissulfosuccinimidyl
suberate (BS3) following the protocol provided by Thermo Scientific. Cell-free crude extracts of
B. muhlenbergius and purified poly-His tagged ORF811 were buffer exchanged into 0.1 M MES
buffer (2-[N-morpholino]ethane sulfonic acid, pH 4.94) using an Econo-Pac 10 DG gel filtration
column. This mixture was cross-linked with 1-ethyl-3-[3-dimethylaminopropyl]carbodiimide
hydrochloride (EDC) following the protocol provided by Thermo Scientific.
Briefly, 10mg EDC was added to 1 mL of ultra-pure (miliQ) H2O to prepare the
crosslinking solution. 100 uL of EDC solution was added to a 1 mL sample of combined protein
solution and incubated at room temperature for two hours. The reaction was desalted by use of
Amicron 4.0 mL Ultra Filtration with addition of MES buffer, centrifuged 3x at 7100xg for 10
min.
For BS3, a 12.5 mM solution was prepared in a 50-molar excess to protein concentration
by adding 277 uL of 20 mM Potassium Phosphate buffer to 2 mg of BS 3. 100 uL of BS3 solution
was added to 500 uL of combined protein sample and incubated at room temperature for 30
min. Tris-HCl buffer was added to quench the reaction and the quenched reaction was stored at
-20°C.
8. SDS-PAGE and Western Blot Analysis
Pull-down and crosslinking samples were analyzed by SDS-PAGE Gel Electrophoresis.
Samples were run on two 4-20% polyacrylamide gels. The gel containing pull-down samples was
stained using GelCode stain while the gel containing the crosslinking samples was
electroblotted to a membrane. The membrane was subjected to staining with Anti-His HRP
Conjugate antibodies.
9. Results:
Bioinformatics Analysis of Orf811
In order to glance at Orf811’s possible structure and function, a bioinformatics approach
was used to find homologs. The fragment of sequenced genome was translated using ExPASy
and run through BLASTP which showed a high relation to Glycine Oxidase from Bacillus subtilis
(Max Score: 447, E-value: 2e-156, top hit). This homolog is ThiO, an FAD dependent oxidase,
and is implicated in thiamine biosynthesis by its close proximity to ThiS, a sulfur carrier, and
ThiF, which is involved in thiamine/molybdopterin sytnthesis. Structural data shows it exists as
a homotetramer (3). Based on its sequence similarity, it is likely that Orf811 will be similar in
function to ThiO.
Preparation and Purification of Bacillus muhlenbergius Cell Lysate
Cultures of B. muhlenbergius were harvested at OD600 = 0.6 and lysed via BeadBeating to
obtain CFCE. Cellular protein was purified to 12.46 mg/mL and stored with glycerol at -20°C.
Protein samples were aliquoted in 800uL amounts for future use. Although the presence of
proteins involved in thiamine biosynthesis was unable to be determined, dark bands close to
the molecular weights of involved proteins (ThiS: 7.89 kDa, ThiG, 26.9 kDa) suggest an increase
in expression of these enzymes.
Pull Down Assay
A pull-down assay involving His-tagged Orf811 was performed to locate stable
protein:protein interactions within the thiamine biosynthetic pathway. SDS-PAGE analysis of all
samples, including flow through and positive controls was performed to view any bound
proteins. Control samples consisted of Bait Control (BC) with no prey protein solution added to
the column and Resin Control (RC) with no Orf811 bound to the column. Flow thru after the
addition of both bait and prey proteins were collected for analysis. Gel bands for experimental
samples were identical to that of bait control (fig. 1), indicating that no stable interactions were
present. The double band shown for Orf811 is consistent with an addition of a small protein;
however, with no difference between sample and control lanes, no additional proteins seem to
10. have been purified. Due to lack of binding, gel bands were not excised for peptide mass
fingerprinting.
11. Cross-Linking Studies Cross-linking studies involving a protein mix of Orf811 and CFCE
were performed using BS3 and EDC. In order to perform these studies, protein solutions were
buffer exchanged into KPO3 monobasic buffer to avoid cross-linker interactions with solvent.
The cross-linked solutions were analyzed via western blotting using His-tag specific antibodies
to observe any changes in molecular weight of Orf811. No bands were observed for EDC cross-
linking, but two additional bands were present for BS3 (fig. 2). Both bands are higher in
molecular weight than Orf811 but do not correspond to an addition of 7.2 kDa (ThiS) or 26.9
kDa (ThiG) (Settembre et al 2004). Molecular weights are consistent with dimer and trimer
formation of GO due to the even spacing of gel bands and their apparent increase in molecular
weight of 47 kDa.
12. Discussion:
We present here an attempt to identify stable and transient protein:protein interactions
between the homotetrameric protein, ThiO and other enzymes in the thiamin biosynthesis
pathway. Although ThiO is known to catalyze the FAD dependent oxidation of glycine to imino-
glycine in this pathway (Settembre et al 2003), several components of the progression remain
unsolved. One unsolved component of this pathway includes the highly reactive imine product
of ThiO; although this product is readily hydrolyzed, the next reaction in the pathway requires
the non-hydrolyzed imine to occur. This reaction, which includes the binding of both
iminoglycine and ThiS to the active site of ThiG, poses the question as to how ThiS and
iminoglycine reach ThiG (Mörtl et al 2004). Since the active site of ThiO is known to be solvent
exposed (Mörtl et al 2004), it is possible that the imine product is transferred to the active site
of ThiG through intermediate shuffling to prevent hydrolysis of iminoglycine (Settembre et al
2004). Analysis of the protein:protein interactions between ThiO and other enzymes in the
thiamin biosynthesis pathway, such as ThiS, may aid in elucidating this potential intermediate
shuffling and the further mapping of the pathway mechanisms. This work includes a pull-down
assay and cross-linking studies to study both stable and transient protein:protein interactions,
respectively.
The newly discovered species of bacteria, Bacillus muhlenbergius, was used in this study.
Prior characterization of this species has revealed a novel enzyme, ORF811, which was used in
all experiments. By utilizing the Basic Local Alignment Search Tool, we found that closely
related homologs of ORF811 were annotated as glycine oxidases, indicating that ORF811 may
also function as a glycine oxidase. Using the BRENDA enzyme database, it was found that a
glycine oxidation reaction catalyzed by an ORF811 homolog consists of glycine, water, and
molecular oxygen reacting to form glycoxylate, ammonia, and hydrogen peroxide; this provides
some insight as to what type of reaction ORF811 may catalyze. Additionally, a genomic context
analysis of closely related homologs of ORF811 revealed that the homologous gene ThiO, as
13. well as its surrounding genes, appears to function in thiamine biosynthesis; this indicates that
ORF811 may play similar roles in metabolism. Thus, the apparent close homology between
ORF811 and ThiO allowed us to use recombinant His-tagged ORF811 to facilitate the study of
protein:protein interactions between ThiO and other enzymes in the thiamin biosynthesis
pathway.
The study was initiated by growing B. muhlenbergius in nutrient deficient media. This
forced the bacteria to produce large amounts of their own thiamin, as well as the proteins
implicated in thiamin biosynthesis, ideally to improve binding of ORF811 to its potential
partners in the pathway. Growth and harvesting of B. muhlenbergius was successful, and 9.5 ml
of cell lysate was recovered from 2 L of culture; a Bradford assay revealed the concentration of
this CFCE to be 12.46 mg/ml.
To study any stable protein:protein interactions, a pull-down assay was conducted,
followed by an SDS-polyacrylamide gel to analyze results. By immobilizing the recombinant His-
tagged ORF811 bait to a column with a cobalt chelate resin, we were able to run a sample of B.
muhlenbergius cell-free crude extract through the column to determine if any proteins in the
extract bound to the immobilized ORF811. However, an SDS-PAGE gel of the eluant revealed no
difference between the bait control, which contained only ORF811 bound to the resin, and the
experimental sample, which contained both the ORF811 bait and the CFCE prey (Fig. 1). Thus,
no stable protein-protein interactions between this close homolog of ThiO and other enzymes
in the thiamin biosynthesis pathway were observed.
Cross-linking studies allowed any transient protein:protein interactions with ORF811 to
be assessed. Following the cross-linking reaction, a Western blot was performed to compare
the size of the band to a control sample of just ORF811; an increase in size would indicate that
binding may have occurred. A solution of ORF811 and B. muhlenbergius CFCE were cross-linked
with both BS3 and EDC. When EDC was used as the cross-linker, no signal was observed on the
Western blot. Prior to the cross-linking reaction, the solution of CFCE and ORF811 was buffer
exchanged three times, which resulted in a significant decrease in protein concentration. Thus,
it is possible that the concentration of protein was too low for any cross-linking to be visualized
14. on the blot. Another EDC cross-linking reaction with a higher concentration of protein is needed
to confirm this conclusion. Although bands were observed when BS 3 was used as the cross-
linker, it is not likely that these bands correspond to any relevant protein:protein interactions
with ThiO. Previous studies have indicated that ThiO is a 47 kDa protein (Job et. al. 2001), and
the three bands on the Western blot (Fig. 2) appear to be equally spaced apart by about 47
kDa; the bands are about 47 kDa, 94 kDa, and 141 kDa. This 47 kDa spacing between bands
suggests that ORF811 formed a homodimer, and is thus not indicative of any relevant transient
protein:protein interactions. However, this conclusion needs to be confirmed by protein
sequencing of the bands.
Although no stable or transient protein:protein interactions with B. muhlenbergius
ORF811 and other enzymes involved in thiamin biosynthesis were observed, further research is
needed to investigate these possible interactions. Addition of a glycine or sulfite substrate may
facilitate the close relation of the possible binding partners, such as ThiS, and thus aid in
assessing these interactions. Further elucidation of the method in which iminoglycine and ThiS
are passed to the active site of ThiG may provide a potential antibiotic target for pathogens that
are susceptible to antibiotics that target thiamin synthesis, such as P. falciparum, the infections
agent responsible for Malaria (Muller et al 2010).
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