RESUMO
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.
Assuntos
Fator de Crescimento Epidérmico/biossíntese , Fator de Crescimento Epidérmico/genética , Produtos do Gene env/biossíntese , Produtos do Gene env/genética , HIV-1/genética , Animais , Western Blotting/métodos , Clonagem Molecular/métodos , Eletroforese em Gel de Poliacrilamida/métodos , Fator de Crescimento Epidérmico/isolamento & purificação , Produtos do Gene env/isolamento & purificação , Vetores Genéticos , Humanos , Fator de Acasalamento , Camundongos , Peptídeos/genética , Pichia , Plasmídeos , Proteínas Recombinantes de Fusão/biossíntese , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/isolamento & purificação , Saccharomyces cerevisiae , Transformação GenéticaRESUMO
Yeasts are attractive hosts for the production of heterologous proteins. Unlike prokaryotic systems, their eukaryotic subcellular organization enables them to carry out many of the post-translational folding, processing and modification events required to produce "authentic" and bioactive mammalian proteins. In addition, they retain the advantages of a unicellular microorganism, with respect to rapid growth and ease of genetic manipulation. The vast majority of yeast expression work has focused on the well-characterized baker's yeast Saccharomyces cerevisiae. However, with the development of DNA transformation technologies, a growing number of non-Saccharomyces yeasts are becoming available as hosts for recombinant polypeptide production. These include Hansenula polymorpha, Kluyveromyces lactis, Pichia pastoris, Schizosaccharomyces pombe, Schwanniomyces occidentalis and Yarrowia lipolytica. The performance of these alternative yeast expression systems is reviewed here relative to S. cerevisiae, and the advantages and limitations of these systems are discussed.