Document 0923
 DOCN  M9440923
 TI    The intracellular production and secretion of HIV-1 envelope protein in
       the methylotrophic yeast Pichia pastoris.
 DT    9404
 AU    Scorer CA; Buckholz RG; Clare JJ; Romanos MA; Wellcome Research
       Laboratories, Beckenham, Kent, UK.
 SO    Gene. 1993 Dec 22;136(1-2):111-9. Unique Identifier : AIDSLINE
       MED/94123987
 AB    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.
 DE    Amino Acid Sequence  Base Sequence  Cloning, Molecular  DNA, Recombinant
       Genetic Vectors  HIV Envelope Protein gp120/*GENETICS  *HIV-1  Molecular
       Sequence Data  Pichia/*GENETICS  Recombinant Proteins/GENETICS/SECRETION
       Support, Non-U.S. Gov't  Transcription, Genetic  JOURNAL ARTICLE

       SOURCE: National Library of Medicine.  NOTICE: This material may be
       protected by Copyright Law (Title 17, U.S.Code).