Purification of hepatitis B surface antigen virus-like particles from recombinant Pichia pastoris and in vivo analysis of their immunogenic properties.

Following earlier studies on high-level intracellular production of hepatitis B surface antigen (HBsAg) using recombinant Pichia pastoris, we present here in detail an enhanced method for the purification of recombinant HBsAg virus-like particles (VLPs). We have screened various detergents for their ability to promote the solubilization of recombinant intracellular HBsAg. In addition, we have analyzed the effect of cell disruption and extraction regarding their impact on the release of HBsAg. Our results show that introduction of the mild nonionic detergent Tween 20 in the initial process of cell lysis at ∼600bars by high pressure homogenization leads to the best results. The subsequent purification steps involved polyethylene glycol precipitation of host cell contaminants, hydrophobic adsorption of HBsAg to colloidal silica followed by ion-exchange chromatography and either isopycnic density ultracentrifugation or size exclusion chromatography for the recovery of the VLPs. After final KSCN treatment and dialysis, a total yield of ∼3% with a purity of >99% was reached. The pure protein was characterized by electron microscopy, showing the presence of uniform VLPs which are the pre-requisite for immunogenicity. The intramuscular co-administration of HBsAg VLPs, with either alum or a PEGylated-derivative of the toll-like receptor 2/6 agonist MALP-2, to mice resulted in the elicitation of significantly higher HBsAg-specific IgG titers as well as a stronger cellular immune response compared to mice vaccinated with a gold standard vaccine (Engerix™). These results show that P. pastoris derived HBsAg VLPs exhibit a high potential as a superior biosimilar vaccine against hepatitis B.

Fed-batch cultivation of Escherichia coli expressed designer hepatitis C virus diagnostic intermediate and its evaluation.

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 ß-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.