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The expression of a hybrid gene encoding an alpha-factor prepro leader peptide-miniproinsulin (MPI) fusion [MPI is the same as the LysArg human insulin precursor described by Thim et al. (1986)] was tested in a series of isogenic yeast strains to investigate the influence of some genetic and physiological factors on heterologous production in yeast. We found that: (i) an MF alpha 1 gene disruption in haploid cells, as well as MF alpha 1 gene product expression in diploid cells, do not affect the MPI secretion level; (ii) under conditions of exogenous leucine availability, MPI production is hindered by leucine auxotrophy (a leu2 mutation); (iii) rho- mutations increase the per-cell MPI yield approximately three-fold; (iv) the MPI yield is apparently dependent on the pH of the culture medium: the higher the external pH, the larger the per-cell MPI yield.

DNA sequences of the envelope (env) gene of the bovine leukemia virus (BLV) were expressed in the yeast Saccharomyces cerevisiae. Two yeast promoters, the repressible PHO5 promoter and the constitutive PGK promoter, were used to construct four expression plasmids comprising either a sequence of the surface antigen gp51 or a (gp51 + gp30) sequence. The expressed heterologous gene products were characterized by Western blot analysis and competitive radioimmunoassay. By means of Northern blot analysis the steady-state level of env-specific mRNA was analysed. The highest expression rate was obtained from recombinant plasmid YEpSG 94 comprising a gp51 sequence--a 630 base pair fragment containing 70% of the gp51 but lacking the N terminus--as well as the PHO5 promoter including PHO5 signal sequence and the PHO5 terminator. The recombinant gp51 was partially glycosylated but the PHO5 signal peptide did not seem to be cleaved off. No immunoreactive material could be found in the periplasm or in the culture medium. By means of monoclonal antibodies directed against eight different epitopes of viral gp51, all four sequential antigenic determinants were detected in the AH 216 (YEpSG 94) expression product.