title: The intracellular production and secretion of HIV-1 envelope protein in the methylotrophic yeast Pichia pastoris.
abstract: The human immunodeficiency virus type 1 (HIV-1) envelope glycoprotein, gp120 (ENV), is required in large quantities for immunological studies and as a potential vaccine component. We have expressed the DNA encoding gp120 in a highly efficient expression system based on the methylotrophic yeast, Pichia pastoris. The native gene was found to contain a sequence which resembled a Saccharomyces cerevisiae polyadenylation consensus and acted as a premature polyadenylation site in P. pastoris, resulting in the production of truncated mRNA. As full-length mRNA was produced in S. cerevisiae, this indicates differences in mRNA 3'-end formation between the two yeasts. Inactivation of this site by site-directed mutagenesis revealed several additional fortuitous polyadenylation sites within the gene. We have designed and constructed a 69%-synthetic gene with increased G + C content which overcomes this transcriptional problem, giving rise to full-length mRNA. High levels of intracellular, insoluble, unglycosylated ENV were produced [1.25 mg/ml in high-density (2 x 10(10) cells per ml) fermentations]. ENV also was secreted from P. pastoris using the S. cerevisiae alpha-factor prepro secretion leader and the S. cerevisiae invertase signal sequence. However, a high proportion of the secreted product was found to be hyperglycosylated, in contrast to other foreign proteins secreted from P. pastoris. There also was substantial proteolytic degradation, but this was minimized by maintaining a low pH on induction. Insoluble, yeast-derived ENV proteins are being considered as vaccine antigens and the P. pastoris system offers an efficient method of production.
authors.affiliation: Wellcome Research Laboratories, Beckenham, Kent, UK.
authors.forename: C A
authors.initials: CA
authors.lastname: Scorer
authors.forename: R G
authors.initials: RG
authors.lastname: Buckholz
authors.forename: J J
authors.initials: JJ
authors.lastname: Clare
authors.forename: M A
authors.initials: MA
authors.lastname: Romanos
mesh_terms: D000595:Amino Acid Sequence; D001483:Base Sequence; D003001:Cloning, Molecular; D004274:DNA, Recombinant; D005822:Genetic Vectors; D015699:HIV Envelope Protein gp120 / Q000235:genetics*; D015497:HIV-1; D008969:Molecular Sequence Data; D010843:Pichia / Q000235:genetics*; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D014158:Transcription, Genetic
chemical_list: D004274:DNA, Recombinant; D015699:HIV Envelope Protein gp120; D011994:Recombinant Proteins