Expression of EGF and HIV envelope glycoprotein.
1. The S. cerevisiae alpha-factor prepro leader is functional and is correctly processed in P. pastoris. 2. P. pastoris has a high secretory capacity, but yields can be severely reduced by extracellular proteases. This problem can be reduced by altering the medium composition, e.g., adjusting the pH or by adding casamino acids. 3. A rapid DNA dot-blot technique can be used for mass screening of transformants to obtain high-copy-number, high-expressing strains. 4. For mEGF, which is an efficiently secreted protein, there was a good correlation between gene dosage and yield, and maximum levels were obtained at high copy number. 5. Vectors conferring resistance to G418 have been developed for the selection of high-copy-number transformants. These vectors can also be used to isolate a series of transformants with increasing copy number of optimizing the expression of genes where high copy number may be detrimental. 6. The HIV-1 ENV gene was not expressed in P. pastoris owing to fortuitous termination of transcription within AT-rich regions. This is a species-specific phenomenon, since full-length HIV-1 ENV transcripts are produced in S. cerevisiae. The problem was overcome by synthesizing the relevent portion of the gene with increased GC content. 7. ENV was hyperglycosylated and immunologically inactive when secreted by P. pastoris. The yield was reduced by extracellular proteases, but like mEGF, this could be significantly improved by altering the pH of the culture medium and by adding casamino acids. 8. In single-copy integrants, transcripts from the semisynthetic HIV-1 ENV gene were almost as abundant as endogenous AOX1. Transcript levels increased progressively with increasing copy number, showing that the AOX1 promoter is not greatly limited by the level of trans-activating factors.
Department of Food Science and Technology, University of Nebraska, Lincoln, NE.
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J
Clare
C
C
Scorer
R
R
Buckholz
M
M
Romanos
D000818:Animals; D015153:Blotting, Western / Q000379:methods; D003001:Cloning, Molecular / Q000379:methods; D004591:Electrophoresis, Polyacrylamide Gel / Q000379:methods; D004815:Epidermal Growth Factor / Q000096:biosynthesis* / Q000235:genetics* / Q000302:isolation & purification; D015686:Gene Products, env / Q000096:biosynthesis* / Q000235:genetics* / Q000302:isolation & purification; D005822:Genetic Vectors; D015497:HIV-1 / Q000235:genetics*; D006801:Humans; D000072235:Mating Factor; D051379:Mice; D010455:Peptides / Q000235:genetics; D010843:Pichia; D010957:Plasmids; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000302:isolation & purification; D012441:Saccharomyces cerevisiae; D014170:Transformation, Genetic
D015686:Gene Products, env; D010455:Peptides; D011993:Recombinant Fusion Proteins; D011994:Recombinant Proteins; D000072235:Mating Factor; D004815:Epidermal Growth Factor