scholarly journals Expression and Purification of the Recombinant Lethal Factor of Bacillus anthracis

1998 ◽  
Vol 66 (2) ◽  
pp. 862-865 ◽  
Author(s):  
Pankaj Gupta ◽  
Smriti Batra ◽  
Arun P. Chopra ◽  
Yogendra Singh ◽  
Rakesh Bhatnagar
Keyword(s):  
Bacillus Anthracis ◽  
Protective Antigen ◽  
Polyacrylamide Gel Electrophoresis ◽  
Lethal Factor ◽  
Sodium Dodecyl ◽  
Gel Filtration ◽  
Nitrilotriacetic Acid ◽  
Biologically Active ◽  
Liquid Chromatograph ◽  
E Coli

ABSTRACT The structural gene for the 90-kDa lethal factor (LF) isolated fromBacillus anthracis was expressed as a fusion protein with six histidine residues in Escherichia coli. Expression of LF in E. coli under the transcriptional regulation of the T5 promoter yielded a soluble cytosolic protein with an apparent molecular mass of 90 kDa, as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Recombinant LF reacted with anti-LF antibodies. The protein was purified to homogeneity by nickel nitrilotriacetic acid affinity chromatography and gel filtration on a Sephacryl S-200 column followed by anion exchange on a fast-performance liquid chromatograph with a Resource-Q column. The yield of purified LF from this procedure was 1.5 mg/liter. In solution, trypsin cleaved protective antigen bound to native and recombinant LF with comparable affinities. In macrophage lysis assays, native and recombinant LF exhibited identical potencies. The results suggest that large amounts of biologically active LF can be purified by this procedure.

scholarly journals Glycerol Monolaurate Inhibits Virulence Factor Production in Bacillus anthracis

2005 ◽  
Vol 49 (4) ◽  
pp. 1302-1305 ◽  
Author(s):  
Sara M. Vetter ◽  
Patrick M. Schlievert
Keyword(s):  
Bacillus Anthracis ◽  
Protective Antigen ◽  
Polyacrylamide Gel Electrophoresis ◽  
Lethal Factor ◽  
Sodium Dodecyl ◽  
The United States ◽  
Human Populations ◽  
Transcriptional Level ◽  
Mrna Levels ◽  
Glycerol Monolaurate

ABSTRACT Anthrax, caused by Bacillus anthracis, has been brought to the public's attention because of the 2001 bioterrorism attacks. However, anthrax is a disease that poses agricultural threats in the United States as well as human populations in Europe, China, Africa, and Australia. Glycerol monolaurate (GML) is a compound that has been shown to inhibit exotoxin production by Staphylococcus aureus and other gram-positive bacteria. Here, we study the effects of GML on growth and toxin production in B. anthracis. The Sterne strain of B. anthracis was grown to post-exponential phase with 0-, 10-, 15-, or 20-μg/ml concentrations of GML and then assayed quantitatively for protective antigen (PA) and lethal factor (LF). After 8 h, GML at concentrations greater than 20 μg/ml was bacteriostatic to growth of the organism. However, a 10-μg/ml concentration of GML was not growth inhibitory, but amounts of PA and LF made were greatly reduced. This effect was not global for all proteins when total secreted protein from culture fluids was examined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Through quantitative reverse transcription-PCR assays, this toxin-inhibitory effect was shown to occur at the transcriptional level, since amounts of mRNA for pagA (PA), lef (LF), and cya (edema factor) were reduced. Surprisingly, mRNA levels of atxA, a regulator of exotoxin gene expression, rose in the presence of GML. These data will be useful in developing therapeutic tools to treat anthrax disease, whether in animals or humans. These results also suggest that mechanisms of virulence regulation exist independent of atxA.

scholarly journals Soluble Expression and Characterization of Biologically Active Bacillus anthracis Protective Antigen in Escherichia coli

2016 ◽  
Vol 2016 ◽  
pp. 1-11 ◽  
Author(s):  
Nagendra Suryanarayana ◽  
Vanlalhmuaka ◽  
Bharti Mankere ◽  
Monika Verma ◽  
Kulanthaivel Thavachelvam ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Bacillus Anthracis ◽  
Protective Antigen ◽  
Expression System ◽  
Lethal Factor ◽  
Biologically Active ◽  
Expression Vectors ◽  
Soluble Form ◽  
Small Scale ◽  
E Coli

Bacillus anthracis secretory protein protective antigen (PA) is primary candidate for subunit vaccine against anthrax. Attempts to obtain large quantity of PA from Escherichia coli expression system often result in the formation of insoluble inclusion bodies. Therefore, it is always better to produce recombinant proteins in a soluble form. In the present study, we have obtained biologically active recombinant PA in small scale E. coli shake culture system using three different expression constructs. The PA gene was cloned in expression vectors bearing trc, T5, and T7 promoters and transformed into their respective E. coli hosts. The growth conditions were optimized to obtain maximum expression of PA in soluble form. The expression construct PA-pET32c in DE3-pLysS E. coli host resulted in a maximum production of soluble PA (15 mg L−1) compared to other combinations. Purified PA was subjected to trypsin digestion and binding assay with lethal factor to confirm the protein’s functionality. Biological activity was confirmed by cytotoxicity assay on J774.1 cells. Balb/c mice were immunized with PA and the immunogenicity was tested by ELISA and toxin neutralization assay. This study highlights the expression of soluble and biologically active recombinant PA in larger quantity using simpler E. coli production platform.

scholarly journals Interactions of an Essential Bacillus subtilis GTPase, YsxC, with Ribosomes

2007 ◽  
Vol 190 (2) ◽  
pp. 681-690 ◽  
Author(s):  
Catherine Wicker-Planquart ◽  
Anne-Emmanuelle Foucher ◽  
Mathilde Louwagie ◽  
Robert A. Britton ◽  
Jean-Michel Jault
Keyword(s):  
Bacillus Subtilis ◽  
Ribosomal Proteins ◽  
Ribosome Biogenesis ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Gel Filtration ◽  
Small Gtpase ◽  
E Coli ◽  
Protein Partners ◽  
The One

ABSTRACT YsxC is a small GTPase of Bacillus subtilis with essential but still unknown function, although recent works have suggested that it might be involved in ribosome biogenesis. Here, purified YsxC overexpressed in Escherichia coli was found to be partly associated with high-molecular-weight material, most likely rRNA, and thus eluted from gel filtration as a large complex. In addition, purification of ribosomes from an E. coli strain overexpressing YsxC allowed the copurification of the YsxC protein. Purified YsxC was shown to bind preferentially to the 50S subunit of B. subtilis ribosomes; this interaction was modulated by nucleotides and was stronger in the presence of a nonhydrolyzable GTP analogue than with GTP. Far-Western blotting analysis performed with His6-YsxC and ribosomal proteins separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed that YsxC interacted with at least four ribosomal proteins from the 50S subunit. Two of these putative protein partners were identified by mass spectrometry as L1 and L3, while the third reactive band in the one-dimensional gel contained L6 and L10. The fourth band that reacted with YsxC contained a mixture of three proteins, L7/L12, L23, and L27, suggesting that at least one of them binds to YsxC. Coimmobilization assays confirmed that L1, L6, and L7/L12 interact with YsxC. Together, these results suggest that YsxC plays a role in ribosome assembly.

scholarly journals Purification of Anthrax Edema Factor fromEscherichia coli and Identification of Residues Required for Binding to Anthrax Protective Antigen

2001 ◽  
Vol 69 (10) ◽  
pp. 6532-6536 ◽  
Author(s):  
Praveen Kumar ◽  
Nidhi Ahuja ◽  
Rakesh Bhatnagar
Keyword(s):  
Recombinant Protein ◽  
Protective Antigen ◽  
Metal Chelate ◽  
Lethal Factor ◽  
Exchange Chromatography ◽  
Biologically Active ◽  
E Coli ◽  
Edema Factor ◽  
Step Procedure ◽  
Anthrax Protective Antigen

ABSTRACT The structural gene for anthrax edema factor (EF) was expressed in Escherichia coli under the control of a powerful T5 promoter to yield the 89-kDa recombinant protein that reacted with anti-EF antibodies. Recombinant EF was purified to homogeneity by a two-step procedure involving metal chelate affinity chromatography and cation-exchange chromatography. From 1 liter of culture, 2.5 mg of biologically active EF was easily purified. This is the first report of purification of anthrax EF from E. coli. EF purified from E. coli was biologically and functionally as active as its Bacillus anthracis counterpart. The recombinant protein could compete with lethal factor for binding to protective antigen. Sequence analysis revealed a stretch of seven amino acids, Val Tyr Tyr Glu Ile Gly Lys, present both in EF (residues 136 to 142) and lethal factor (residues 147 to 153). To investigate the role of these seven residues in binding to protective antigen, the residues were individually mutated to alanine in EF. Mutations in residues Tyr137, Tyr138, Ile140, and Lys142 of EF specifically blocked its interaction with anthrax protective antigen. The adenylate cyclase activity of the mutants remained unaffected. The results suggested that residues Tyr137, Tyr138, Ile140, and Lys142 are required for binding of EF to anthrax protective antigen, which facilitates its entry into susceptible cells.

Fermentation, Purification, and Characterization of Protective Antigen from a Recombinant, Avirulent Strain ofBacillus anthracis

1998 ◽  
Vol 64 (3) ◽  
pp. 982-991 ◽  
Author(s):  
J. W. Farchaus ◽  
W. J. Ribot ◽  
S. Jendrek ◽  
S. F. Little
Keyword(s):  
Gel Electrophoresis ◽  
Protective Antigen ◽  
Polyacrylamide Gel Electrophoresis ◽  
Lethal Factor ◽  
Sodium Dodecyl ◽  
The United States ◽  
Reversed Phase ◽  
Avirulent Strain ◽  
Purification And Characterization

ABSTRACT Bacillus anthracis, the etiologic agent for anthrax, produces two bipartite, AB-type exotoxins, edema toxin and lethal toxin. The B subunit of both exotoxins is an M r83,000 protein termed protective antigen (PA). The human anthrax vaccine currently licensed for use in the United States consists primarily of this protein adsorbed onto aluminum oxyhydroxide. This report describes the production of PA from a recombinant, asporogenic, nontoxigenic, and nonencapsulated host strain of B. anthracis and the subsequent purification and characterization of the protein product. Fermentation in a high-tryptone, high-yeast-extract medium under nonlimiting aeration produced 20 to 30 mg of secreted PA per liter. Secreted protease activity under these fermentation conditions was low and was inhibited more than 95% by the addition of EDTA. A purity of 88 to 93% was achieved for PA by diafiltration and anion-exchange chromatography, while greater than 95% final purity was achieved with an additional hydrophobic interaction chromatography step. The purity of the PA product was characterized by reversed-phase high-pressure liquid chromatography, sodium dodecyl sulfate (SDS)-capillary electrophoresis, capillary isoelectric focusing, native gel electrophoresis, and SDS-polyacrylamide gel electrophoresis. The biological activity of the PA, when combined with excess lethal factor in the macrophage cell lysis assay, was comparable to previously reported values.

scholarly journals Purification and Characterization of Turnip Mosaic Virus Helper Component Protein

1999 ◽  
Vol 89 (7) ◽  
pp. 564-567 ◽  
Author(s):  
R. Y. Wang ◽  
T. P. Pirone
Keyword(s):  
Molecular Mass ◽  
Mosaic Virus ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Gel Filtration ◽  
Turnip Mosaic Virus ◽  
Biologically Active ◽  
Helper Component ◽  
Pass Through

The helper component (HC) protein of turnip mosaic virus (TuMV) was concentrated by differential centrifugation followed by ammonium sulfate precipitation. The partially purified HC was then loaded onto a Ni2+-resin column that bound the HC; a histidine tag was not required for binding. The HC eluted from the column migrated as a band of about 50 kDa in sodium dodecyl sulfate-polyacrylamide gel electrophoresis. In its native state, the HC did not pass through an ultrafiltration membrane with a molecular mass cutoff of 100 kDa, which suggested that the HC is in a multimeric form when it is biologically active. The molecular mass of the multimeric form was determined by gel filtration to be approximately 145 kDa. Purified HC retained its activity for several months at -20°C. Using a protein blotting-overlay protocol, purified HC interacted in vitro with an aphid-transmissible TuMV isolate, but not with a non-aphid-transmissible isolate.

scholarly journals Restoration of β-galactosidase to Escherichia coli M15. Complementation studies

1977 ◽  
Vol 165 (3) ◽  
pp. 417-423 ◽  
Author(s):  
Dobrivoje V. Marinkovic ◽  
Jelka N. Marinkovic
Keyword(s):  
Escherichia Coli ◽  
Sodium Dodecyl Sulphate ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Gel Filtration ◽  
Galactosidase Activity ◽  
Terminal Amino Acid ◽  
E Coli ◽  
Molecular Weights ◽  
Terminal Amino

Carboxymethylated β-galactosidase from Escherichia coli was dissociated at 100°C to form carboxymethylated fragments A and B. The mol.wts. of carboxymethylated fragments A and B were determined by gel filtration to be 64300 and 22400 respectively. Sodium dodecyl sulphate/polyacrylamide-gel electrophoresis of carboxymethylated fragments A and B that had been pretreated with 2-mercaptoethanol and sodium dodecyl sulphate yielded mol.wts. of 64000 and 22100 respectively. Carboxymethylated fragments A and B had arginine as their C-terminal amino acid. When a crude extract of E. coli M15 was filtered through a column of Sepharose 6B, it was found that carboxymethylated fragment B could restore β-galactosidase activity when added to fractions having mol.wts. estimated to be 123000, 262000 and 506000. These fractions are referred to as ‘complementable fractions’. Similarly, it was found that carboxymethylated fragment A could restore enzyme activity to tractions having mol.wts. estimated to be 63000, 253000 and 506000. Estimates of the molecular weights of the β-galactosidase activity obtained by restoration with carboxymethylated fragments A and B were made by filtering the active enzyme through another column of Sepharose 6B. The enzyme obtained by complementation with carboxymethylated fragment B, i.e. the complemented enzyme, had mol.wt. 525000, and that obtained with carboxymethylated fragment A had mol.wts. of 525000, 646000 and 2000000. The latter finding suggests that multiple forms of complemented β-galactosidase can exist.

scholarly journals Production and Purification of Protective Antigen of Bacillus anthracis and Development of a Sandwich ELISA for its Detection

2020 ◽  
Vol 5 (4) ◽  
pp. 299-304
Author(s):  
Nidhi Puranik ◽  
Manoj Kumar ◽  
Nagesh K Tripathi ◽  
Vijai Pal ◽  
Ajay Goel
Keyword(s):  
Bacillus Anthracis ◽  
Protective Antigen ◽  
Sandwich Elisa ◽  
Lethal Factor ◽  
Gel Filtration ◽  
Point Of View ◽  
Detection Sensitivity ◽  
Diagnostic Systems ◽  
Edema Factor ◽  
Potential Biomarker

Anthrax, a zoonotic disease caused by Bacillus anthracis is important for biowarfare as well as public health point of view. The virulence factors of B. anthracis are encoded by the two plasmids, pXO1 and pXO2. Protective antigen (PA), an 83 kDa protein encoded by pXO1 along with lethal factor (LF, 90 kDa) or edema factor (EF, 89 kDa), makes the anthrax toxin responsible for causing the disease. Current detection and diagnostic systems for anthrax are mostly based on PA, a potential biomarker of B. anthracis. The objective of the present study was to produce and purify the PA for development of a sandwich ELISA for its detection. In this study, pYS5 plasmid containing the full PA gene was transformed into an 8 proteases deficient Bacillus anthracis host BH480. The PA was produced under shake flask conditions and purified using the gel filtration chromatography. The reactivity of PA with rabbit and mouse anti-PA antibodies was confirmed by Western blotting. The antibodies were purified and used for the development of a sandwich ELISA for detection of PA. The detection sensitivity of ELISA was found to be 3.9 ng/ mL PA.

scholarly journals Analysis of Soybean Protein-Derived Peptides and the Effect of Cultivar, Environmental Conditions, and Processing on Lunasin Concentration in Soybean and Soy Products

2008 ◽  
Vol 91 (4) ◽  
pp. 936-946 ◽  
Author(s):  
Wenyi Wang ◽  
Vermont P Dia ◽  
Miguel Vasconez ◽  
Elvira Gonzalez de Mejia ◽  
Randall L Nelson
Keyword(s):  
Environmental Factors ◽  
Bioactive Peptides ◽  
Soybean Protein ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Health Benefits ◽  
Gel Filtration ◽  
Exchange Chromatography ◽  
Biologically Active ◽  
Soybean Cultivar

Abstract Soybean, an important source of food proteins, has received increasing interest from the public because of its reported health benefits. These health benefits are attributed to its components, including isoflavones, saponins, proteins, and peptides. Lunasin, Bowman-Birk inhibitor, lectin, and -conglycinin are some of the biologically active peptides and proteins found in soybean. This article provides a comprehensive review on the recently used techniques in the analysis and characterization of food bioactive peptides, with emphasis on soybean peptides. The methods used to isolate and purify lunasin from defatted soybean flour were ion-exchange chromatography, ultrafiltration, and gel filtration chromatography. The identity of lunasin was established by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, Western blot, matrix-assisted laser desorption ionizationtime of flight, and liquid chromatography. The results on the effect of soybean cultivar and environmental factors on lunasin concentration are also reported. The highest lunasin concentration, 11.7 0.3 mg/g flour, was found in Loda soybean cultivar grown at 23C; the lowest concentration, 5.4 0.4 mg/g flour, was found in Imari soybean cultivar grown at 28C. Lunasin concentration was affected by cultivartemperature, cultivarsoil moisture, and cultivartemperaturesoil moisture interactions. The variation on lunasin concentration suggests that its content can be improved by breeding, and by optimization of growing conditions. In summary, bioactive peptides can be accurately identified and quantified by using different techniques and conditions. In addition, lunasin concentration in soybean depends mainly on cultivar and to some extent on environmental factors, particularly temperature. Lunasin concentration in soy products was also affected by processing conditions.

scholarly journals α-Lactalbumin and lactose concentrations in rat milk during lactation

1981 ◽  
Vol 194 (1) ◽  
pp. 149-154 ◽  
Author(s):  
K R Nicholas ◽  
P E Hartmann ◽  
B L McDonald
Keyword(s):  
Molecular Weight ◽  
Gel Electrophoresis ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Gel Filtration ◽  
Significant Negative Correlation ◽  
Ion Exchange Chromatography ◽  
Exchange Chromatography ◽  
Biologically Active ◽  
Alpha Lactalbumin

Homogeneous rat alpha-lactalbumin was prepared from whey by chromatography on DEAE-Sephadex A-50 and Ultrogel AcA 44. Two biologically active forms of alpha-lactalbumin were apparent after ion-exchange chromatography, but on gel filtration the combined forms were eluted as a single peak with a molecular weight of approx. 33000. The molecular weight when determined by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis was 15100. Antiserum to alpha-lactalbumin was prepared from rabbits, and single radial immunodiffusion was used to measure the concentration of alpha-lactalbumin in milk expressed from rats during lactation and for 2 days after the cessation of lactation. A significant positive correlation (r = + 0.89) between the concentrations of alpha-lactalbumin and lactose was obtained for the first 20 days of lactation. This is consistent with the suggestion that alpha-lactalbumin may control the concentration of lactose in milk. However, a significant negative correlation (r = -0.91) between the concentration of alpha-lactalbumin and lactose was obtained for 2 days after the cessation of lactation on day 20.

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