title: Fed-batch cultivation of Escherichia coli expressed designer hepatitis C virus diagnostic intermediate and its evaluation.
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 β-D-1-thiogalactopyranoside (IPTG) for ∼4 H at 30°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 ∼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.
authors.affiliation: Recombinant Gene Products Group, International Centre for Genetic Engineering and Biotechnology, New Delhi, India. chskrg@gmail.com
authors.forename: Chandrasekhar
authors.initials: C
authors.lastname: Gurramkonda
authors.forename: Sheikh M
authors.initials: SM
authors.lastname: Talha
authors.forename: Satheesh Kumar
authors.initials: SK
authors.lastname: Gudi
authors.forename: Venkateswara Rao
authors.initials: VR
authors.lastname: Gogineni
authors.forename: Krothapalli Raja
authors.initials: KR
authors.lastname: Surya Sambasiva Rao
mesh_terms: D000595:Amino Acid Sequence; D000956:Antigens, Viral / Q000276:immunology; D061249:Batch Cell Culture Techniques; D000939:Epitopes / Q000276:immunology; D004926:Escherichia coli / Q000166:cytology / Q000235:genetics* / Q000254:growth & development*; D015870:Gene Expression; D005818:Genetic Engineering / Q000379:methods*; D016174:Hepacivirus / Q000235:genetics* / Q000276:immunology / Q000302:isolation & purification*; D008969:Molecular Sequence Data; D058849:Protein Refolding; D014764:Viral Proteins / Q000737:chemistry / Q000235:genetics* / Q000276:immunology / Q000302:isolation & purification
chemical_list: D000956:Antigens, Viral; D000939:Epitopes; D014764:Viral Proteins