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 anti gen (HBsAg) p24(s) monomer and lipoprotein particle synthesis produced in Saccharomyces cerevisiae using galactose-limited continuous cultur e. 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-mu m plas mid harbored in a haploid yeast strain. Monomers autonomously form lip oprotein aggregates (particles) in vivo using only host-cell-derived c omponents. 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, estima ted at five- to tenfold by mass, was found at all dilution rates. The average copy number of the 2-mu m plasmid (carrying LEU2 selection) re mained constant at 200 copies per cell from washout to 0.035 h(-1). Su rprisingly, the average copy number was undetectable at the lowest dil ution rate tested (0.015 h(-1)), even though HBsAg expression was maxi mal. Total p24(s) monomer and HBsAg particle values ranged twofold ove r this dilution rate range. No differences in the trends for HBsAg exp ression and average copy number could be detected past the critical di lution rate where aerobic fermentation of galactose and ethanol overfl ow were observed. HBsAg expression in continuous culture was stable fo r at least 40 generations at 0.100 h(-1). (C) 1996 John Wiley & Sons, Inc.