A1 Vertaisarvioitu alkuperäisartikkeli tieteellisessä lehdessä
Fed-batch cultivation of Escherichia coli expressed designer hepatitis C virus diagnostic intermediate and its evaluation
Tekijät: Gurramkonda C, Talha SM, Gudi SK, Gogineni VR, Rao KRSS
Kustantaja: WILEY-BLACKWELL
Julkaisuvuosi: 2012
Journal: Biotechnology and Applied Biochemistry
Tietokannassa oleva lehden nimi: BIOTECHNOLOGY AND APPLIED BIOCHEMISTRY
Lehden akronyymi: BIOTECHNOL APPL BIOC
Vuosikerta: 59
Numero: 6
Aloitussivu: 437
Lopetussivu: 444
Sivujen määrä: 8
ISSN: 0885-4513
DOI: https://doi.org/10.1002/bab.1044
Tiivistelmä
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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