Enhanced secretion of human nerve growth factor from Saccharomyces cerevisiae using an advanced delta-integration system.
We have designed an advanced delta-integration system (integration of genes into the delta-sequence of yeast retrotransposon Ty) and used it for secretion of human nerve growth factor (hNGF) from Saccharomyces cerevisiae. The expression and secretion of hNGF was directed by the PGK promoter and MF alpha 1 prepro-signal. Using two selectable markers (URA3 and leu2-d), haploid yeast strains were constructed with approximately 20 copies of a delta-integrated hNGF expression cassette on four chromosomes. The strain secreted hNGF at levels 3-4 fold higher than a 2 micron-based plasmid. Northern and Western analyses revealed that the oversecretion was caused by an increased amount of mRNA. We also detected an unusual processing of the MF alpha 1 prepro-hNGF fusion protein that required the pep4 mutation. Application of this system for industrial purposes is discussed.
Sakai
A
A
Laboratory of Molecular Genetics, Mitsubishi Kasei Institute of Life Sciences, Tokyo, Japan.
Ozawa
F
F
Higashizaki
T
T
Shimizu
Y
Y
Hishinuma
F
F
D001709:Biotechnology; D004251:DNA Transposable Elements; D006801:Humans; D009414:Nerve Growth Factors / Q000235:genetics* / Q000378:metabolism*; D010957:Plasmids; D011401:Promoter Regions, Genetic; D011498:Protein Precursors / Q000235:genetics / Q000378:metabolism; D011499:Protein Processing, Post-Translational; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000235:genetics*
D004251:DNA Transposable Elements; D009414:Nerve Growth Factors; D011498:Protein Precursors; D011993:Recombinant Fusion Proteins