Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:3.2.1.26 (invertase)
 4,927 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

We have developed a vector system for high-copy-number integration into the ribosomal DNA of the yeast Kluyveromyces lactis. This system is analogous to the pMIRY-system developed for Saccharomyces cerevisiae. Plasmids containing a portion of K. lactis rDNA for targeted homologous recombination, as well as the S. cerevisiae TRP1 gene with various promoter deletions, were constructed and, after transformation to K. lactis, analyzed for both copy number and stability. These plasmids were found to be present in about 60 copies per cell and were stably maintained during growth under non-selective conditions. Using this vector system, we expressed a fusion construct containing the S. cerevisiae GAL7 promoter, the SUC2 (invertase) signal sequence and the gene coding for alpha-galactosidase from the plant Cyamopsis tetragonoloba. Although the maximum copy number of these integrated plasmids was only about 15, we nevertheless obtained a high level of alpha-galactosidase production (250 mg/l) with a secretion efficiency of about 95%. When compared to extrachromosomal K. lactis vectors containing the same fusion construct, the multicopy integrants showed a much higher alpha-galactosidase production level and a considerably higher stability under non-selective conditions.
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PMID:Multiple-copy integration of the alpha-galactosidase gene from Cyamopsis tetragonoloba into the ribosomal DNA of Kluyveromyces lactis. 132 15

We have developed an efficient production system for porcine pancreatic phospholipase A2 in Saccharomyces cerevisiae (baker's yeast). The cDNA encoding the prophospholipase A2 was expressed under the control of the galactose inducible GAL7 promotor, and secretion was directed by the secretion signals of yeast invertase. This construct yielded up to 6 mg prophospholipase A2 activity per 1 fermentation broth, secreted as a glycosylated invertase prophospholipase A2 hybrid protein. Upon genetically deleting the glycosylation site, the level of secretion decreased to 3.6 mg prophospholipase A2 per 1. Changing the invertase secretion signals for an invertase/alpha-mating factor prepro sequence-fusion increased the secretion level up to 8 mg per 1. The secreted non-glycosylated prophospholipase A2 species was correctly processed. Our results demonstrate the promises and limitations for rational design to obtain high level expression and secretion of heterologous proteins by S. cerevisiae.
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PMID:The use of genetic engineering to obtain efficient production of porcine pancreatic phospholipase A2 by Saccharomyces cerevisiae. 185 38

For expression of the alpha-galactosidase gene from Cyamopsis tetragonoloba in Kluyveromyces marxianus CBS 6556 we have used the promoter of the homologous inulinase-encoding gene (INU1). The INU1 gene has been cloned and sequenced and the coding region shows an identity of 59% with the Saccharomyces cerevisiae invertase gene (SUC2). In the 5'-flanking region of INU1 we found a sequence (TAAATCCGGGG) that perfectly matches to the MIG1 binding consensus sequence (WWWWTSYGGGG) of the S. cerevisiae GAL1, GAL4 and SUC2 genes. Using the K. marxianus INU1 promoter and prepro-signal sequence, we obtained a high alpha-galactosidase production level (153 mg/l) and a secretion efficiency of 99%. Both the production level and the secretion efficiency were significantly reduced when the INU1 pro-peptide was deleted. With either the S. cerevisiae PGK or GAL7 promoter we could obtain only low alpha-galactosidase production levels (2 mg/l).
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PMID:Expression of an alpha-galactosidase gene under control of the homologous inulinase promoter in Kluyveromyces marxianus. 776 85