Continuous culture study of the expression of hepatitis B surface antigen and its self-assembly into virus-like particles in Saccharomyces cerevisiae.

We have studied the growth rate dependence of hepatitis B surface antigen (HBsAg) p24(s) monomer and lipoprotein particle synthesis produced in Saccharomyces cerevisiae using galactose-limited continuous culture. The hepatitis B virus S gene, which encodes the p24(s) monomer, is transcribed under the control of the GAL 10p on a chimeric 2-microm plasmid harbored in a haploid yeast strain. Monomers autonomously form lipoprotein aggregates (particles) in vivo using only host-cell-derived components. Steady states were evaluated in a range from 0.015 h(-1) to washout (0.143 h(-1)). Both p24(s) monomer and HBsAg particle levels, at steady state, varied in an inverse linear manner with growth rate. A consistent excess of total p24(s) monomer to HBsAg particle, estimated at five- to tenfold by mass, was found at all dilution rates. The average copy number of the 2-microm plasmid (carrying LEU2 selection) remained constant at 200 copies per cell from washout to 0.035 h(-1). Surprisingly, the average copy number was undetectable at the lowest dilution rate tested (0.015 h(-1)), even though HBsAg expression was maximal. Total p24(s) monomer and HBsAg particle values ranged twofold over this dilution rate range. No differences in the trends for HBsAg expression and average copy number could be detected past the critical dilution rate where aerobic fermentation of galactose and ethanol overflow were observed. HBsAg expression in continuous culture was stable for at least 40 generations at 0.100 h(-1).