Browsing publications of the research group recombinant protein expression (RPEX) by Authors
Assessing stability and assembly of the hepatitis B surface antigen into virus-like particles during down-stream processing.Zahid, Maria; Lünsdorf, Heinrich; Rinas, Ursula; Helmholtz Centre for infection research, Inhoffenstr. 7, 38124 Braunschweig, Germany. (2015-07-17)The hepatitis B surface antigen (HBsAg) is a recombinant protein-based vaccine being able to form virus-like particles (VLPs). HBsAg is mainly produced using yeast-based expression systems, however, recent results strongly suggest that VLPs are not formed within the yeast cells during the cultivation but are formed in a gradual manner during the following down-stream procedures. VLPs are also not detectable during the first down-stream steps including mechanical and EDTA/detergent-assisted cell destruction. Moreover, VLPs are not detectable in the cell lysate treated with polyethylene glycol and colloidal silica. The first VLP resembling structures appear after elution of HBsAg from colloidal silica to which it binds through hydrophobic interaction. These first VLP resembling structures are non-symmetrical as well as heterodisperse and exhibit a high tendency toward cluster formation presumably because of surface exposed hydrophobic patches. More symmetrical and monodisperse VLPs appear after the following ion-exchange and size-exclusion chromatography most likely as the result of buffer changes during these purification steps (toward more neutral pH and less salt). Final treatment of the VLPs with the denaturant KSCN at moderate concentrations with following KSCN removal by dialysis does not cause unfolding and VLP disassembly but results in a re- and fine-structuring of the VLP surface topology.
Purification of hepatitis B surface antigen virus-like particles from recombinant Pichia pastoris and in vivo analysis of their immunogenic properties.Gurramkonda, Chandrasekhar; Zahid, Maria; Nemani, Satish Kumar; Adnan, Ahmad; Gudi, Satheesh Kumar; Khanna, Navin; Ebensen, Thomas; Lünsdorf, Heinrich; Guzmán, Carlos A; Rinas, Ursula; et al. (2013-12-01)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.