Fermentation study for the production of hepatitis B virus pre-S2 antigen by the methylotrophic yeastHansenula polymorpha

Abstract

Various physico-chemical parameters have been studied in order to improve the production of hepatitis B virus pre-S2 antigen (middle surface antigen) by the methylotrophic yeast Hansenula polymorpha. Antigen production was done in two steps: first, production of cells on glycerol (Phase 1), followed by induction of antigen expression with methanol (Phase 2). Dense cultures of H. polymorpha, equivalent to 35-40 g/l (dry weight), were readily obtained in small fermenters using minimal medium containing glycerol as carbon source. Antigen expression in this minimal medium, after induction with methanol, was however, low and never exceeded 1.6 mg/l of culture. Antigen production was greatly enhanced by adding complex organic nitrogen sources along with methanol at induction time; yeast extract was the best of all the sources tested. In shake flasks, antigen production was proportional to yeast extract concentration up to 7% (w/v) yeast extract, it became clear the the nutritional conditions for good antigen expression were different from those for good biomass production. The effects of yeast extract were reproduced in small fermenters: antigen levels reached 8-9 mg/l in medium containing 6% (w/v) yeast extract during induction with methanol. The mechanisms of yeast extract's effects are still unknown but are probably nutritional. The recombinant H. polymorpha strain produced both periplasmic and intracellular antigen. The periplasmic antigen was shown to be present as 20-22-nm particles and was therefore immunogenic. Immunoblotting indicated that part of the pre-S2 antigen was present as a 24-kDa degradation product. These studies have led to a 140-fold increase in volumetric productivity of antigen and to a 4.6-fold increase in specific production.

Influence of pH, nitrogen and phosphorus sources on the production of hepatitis B virus pre-S2 antigen by Hansenula polymorpha

“…The recombinant H. polymorpha strain (uracil auxotroph) producing the hepatitis B virus pre-S2 surface antigen has been described previously (de Roubin et al 1991).…”

“…Succinic acid (10 g/l) was used as buffer. Solutions A and B and the vitamin solution were as described previously (de Roubin et al 1991). These solutions were all filter-sterilized (0.2 ~tm).…”

“…The flasks were inoculated as described previously (de Roubin et al 1991) and incubated at 37°C for up to 8 days at an orbital agitation rate of 300rpm (radius of gyration: 2.5 cm). Unless otherwise indicated, antigen production was measured 6 days after inoculation based on earlier observations.…”

“…polymorpha has been used in our laboratory for the experimental production of the hepatitis B virus pre-$2 antigen (middle protein) under control of the methanol oxidase promoter (Shen et al 1989;de Roubin et al 1991de Roubin et al , 1992. This protein has been reported to be a more efficient vaccine than the S antigen (major protein) (Langley et al 1988).…”

“…This protein has been reported to be a more efficient vaccine than the S antigen (major protein) (Langley et al 1988). Appreciable quantities of this antigen were obtained only when very rich media were employed and, in addition, the nature of the nitrogen source was shown to be critical (de Roubin et al 1991). An important observation was that the distribution of the pre-S2 antigen between the periplasm and the cytoplasm of the yeast could be modulated, to a large extent, by adjusting some physical and nutritional factors (de Roubin et al 1992).…”