title: Development of a simple fed-batch process for the high-yield production of recombinant shiga toxin B-chain protein.
abstract: Shiga toxins are one of the very potent agents for causing dysentery, diarrhoea and haemolytic uremic syndrome with very low LD50. For better understanding of their biology, detection and neutralization, the components of toxins are needed to be expressed and purified in bulk amounts. However, following traditional expression procedures, this task is very tedious as the yield of the toxin is very low. In this manuscript, we have described the optimization of media for enhanced production of recombinant Shiga toxin B (rStx-B) chain protein in Escherichia coli. This protein is known to have neutralization ability against shiga toxins. Furthermore, fed-batch cultivation process in E. coli was also developed in the optimized medium. Expression was induced with 1 mM isopropyl-beta-thiogalactoside (IPTG). The purification of protein involved Ni-NTA affinity chromatography under native conditions followed by gel filtration chromatography. After fed-batch cultivation, the recombinant E. coli resulted in cell weight and purified protein of about 19.41 g/l (dry cell weight, 11.38 g/l) and 30 mg/l of culture, respectively. The purity of the recombinant StxB protein was checked by sodium dodecyl sulfate polyacrylamide gel electrophoresis analysis. Reactivity of this protein was determined by Western blotting as well as indirect ELISA using specific antibodies. These results establish the application of this protein for diagnosis of shiga toxin infection or for neutralizing the toxicity.
authors.affiliation: Bioprocess Scale-Up Facility, Defence Research and Development Establishment, Jhansi Road, Gwalior 474002, India. kulsviro@gmail.com
authors.forename: Kulbhushan
authors.initials: K
authors.lastname: Sharma
authors.forename: Pallavi
authors.initials: P
authors.lastname: Gupta
authors.forename: Anil
authors.initials: A
authors.lastname: Kumar Singh
authors.forename: N
authors.initials: N
authors.lastname: Gopalan
authors.forename: Ram
authors.initials: R
authors.lastname: Kumar Dhaked
authors.forename: P V
authors.initials: PV
authors.lastname: Lakshmana Rao
mesh_terms: D061249:Batch Cell Culture Techniques / Q000379:methods*; D019149:Bioreactors; D015153:Blotting, Western; D002846:Chromatography, Affinity; D004526:Efficiency; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000254:growth & development / Q000378:metabolism; D005285:Fermentation / Q000502:physiology; D021122:Protein Subunits / Q000032:analysis / Q000138:chemical synthesis / Q000737:chemistry / Q000302:isolation & purification; D011994:Recombinant Proteins / Q000032:analysis / Q000138:chemical synthesis / Q000737:chemistry / Q000302:isolation & purification; D022621:Shiga Toxin / Q000032:analysis / Q000138:chemical synthesis* / Q000737:chemistry / Q000302:isolation & purification
chemical_list: D021122:Protein Subunits; D011994:Recombinant Proteins; D022621:Shiga Toxin