Efficient production of intact human parathyroid hormone in a Saccharomyces cerevisiae mutant deficient in yeast aspartic protease 3 (YAP3).
When human parathyroid hormone (hPTH) is expressed as a secretory product in yeast, the main problem is the aberrant proteolytic cleavage that reduces the yield of intact protein. To overcome this problem, we developed an hPTH expression system using a host strain in which the YAP3 gene encoding yeast aspartic protease 3 (YAP3) was disrupted. After 48 h of culture, most of the hPTH secreted by the yap3 disruptant remained intact, whereas more than 90% of the hPTH secreted by the wild-type strain was cleaved. When the authentic hPTH was incubated in each of the culture supernatants of untransformed yap3 disruptant and wild-type strain, the proteolysis proceeded much more slowly in the culture supernatant of yap3 disruptant than in that of the wild type. The extent of hPTH proteolysis was also significantly reduced by the addition of pepstatin A, a specific aspartic protease inhibitor. The results suggest that YAP3 is involved in the internal cleavage of hPTH expressed in yeast. The correct processing of the intact hPTH secreted in the yap3 disruptant demonstrates that the yeast mutant lacking the YAP3 activity is a suitable host for the high-level expression of intact hPTH.
Kang
H A
HA
Korea Research Institute of Bioscience and Biotechnology, Taejon, Korea.
Kim
S J
SJ
Choi
E S
ES
Rhee
S K
SK
Chung
B H
BH
D016282:Aspartic Acid Endopeptidases / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D009154:Mutation; D010281:Parathyroid Hormone / Q000096:biosynthesis* / Q000378:metabolism; D010436:Pepstatins / Q000378:metabolism; D010446:Peptide Fragments / Q000378:metabolism; D011480:Protease Inhibitors / Q000378:metabolism; D011499:Protein Processing, Post-Translational; D011994:Recombinant Proteins / Q000096:biosynthesis; D012441:Saccharomyces cerevisiae / Q000201:enzymology* / Q000235:genetics* / Q000254:growth & development; D029701:Saccharomyces cerevisiae Proteins; D014170:Transformation, Genetic
D010281:Parathyroid Hormone; D010436:Pepstatins; D010446:Peptide Fragments; D011480:Protease Inhibitors; D011994:Recombinant Proteins; D029701:Saccharomyces cerevisiae Proteins; C014195:Streptomyces pepsin inhibitor; C022325:aspartic proteinase A; D016282:Aspartic Acid Endopeptidases; C499266:YPS1 protein, S cerevisiae; C031375:pepstatin