A1 Refereed original research article in a scientific journal
Fed-batch cultivation of Escherichia coli expressed designer hepatitis C virus diagnostic intermediate and its evaluation
Authors: Gurramkonda C, Talha SM, Gudi SK, Gogineni VR, Rao KRSS
Publisher: WILEY-BLACKWELL
Publication year: 2012
Journal: Biotechnology and Applied Biochemistry
Journal name in source: BIOTECHNOLOGY AND APPLIED BIOCHEMISTRY
Journal acronym: BIOTECHNOL APPL BIOC
Volume: 59
Issue: 6
First page : 437
Last page: 444
Number of pages: 8
ISSN: 0885-4513
DOI: https://doi.org/10.1002/bab.1044
Abstract
The present study aimed for an enhanced induction strategy combined with high-level production of a capture antigen of hepatitis C virus (HCV) for use in diagnosis of HCV infection. We have expressed the synthetic gene encoding for HCV multiepitope protein in pET-28a(+) vector and investigated its production in Escherichia coli BL21(DE3) cells using high-cell-density fed-batch cultivation. A maximum cell dry mass of 30 g/L was obtained, and the culture was induced with 1, 5, and 10 mM isopropyl beta-d-1-thiogalactopyranoside (IPTG) for similar to 4 H at 30 degrees C; a maximum protein production of 1.5 g/L was observed in the case of induction with 10 mM IPTG. The enhanced induction strategy resulted in a similar to 15-fold increase as compared to 1 mM IPTG. The protein was purified using a simple immobilized metal affinity chromatography procedure, yielding 16.6 mg/g dry cell weight of pure protein with more than 99% purity. Further, the protein was evaluated for its diagnostic potential by using the commercially available HCV Seroconversion Panel, Worldwide HCV Performance Panel, and Viral Coinfection Panel. The protein showed high sensitivity and specificity, which was comparable to the best performing commercially available enzyme immunoassay (EIA) kits.
The present study aimed for an enhanced induction strategy combined with high-level production of a capture antigen of hepatitis C virus (HCV) for use in diagnosis of HCV infection. We have expressed the synthetic gene encoding for HCV multiepitope protein in pET-28a(+) vector and investigated its production in Escherichia coli BL21(DE3) cells using high-cell-density fed-batch cultivation. A maximum cell dry mass of 30 g/L was obtained, and the culture was induced with 1, 5, and 10 mM isopropyl beta-d-1-thiogalactopyranoside (IPTG) for similar to 4 H at 30 degrees C; a maximum protein production of 1.5 g/L was observed in the case of induction with 10 mM IPTG. The enhanced induction strategy resulted in a similar to 15-fold increase as compared to 1 mM IPTG. The protein was purified using a simple immobilized metal affinity chromatography procedure, yielding 16.6 mg/g dry cell weight of pure protein with more than 99% purity. Further, the protein was evaluated for its diagnostic potential by using the commercially available HCV Seroconversion Panel, Worldwide HCV Performance Panel, and Viral Coinfection Panel. The protein showed high sensitivity and specificity, which was comparable to the best performing commercially available enzyme immunoassay (EIA) kits.
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