Multiple-yapsin-deficient mutant strains for high-level production of intact recombinant proteins in Saccharomyces cerevisiae.
The yapsin family of aspartic proteases, located at cell surface, has a common specificity for paired or single basic reside cleavage sites of proproteins. Our previous study reported that the aberrant proteolytic cleavage of secretory recombinant human parathyroid hormone (hPTH) protein was problematic at late stages of fed-batch cultivations, even in the Saccharomyces cerevisiae mutant strain deficient in yapsin 1 (yps1Delta). To overcome this problem, we constructed a set of S. cerevisiae mutant strains lacking several members of the yapsin family through disruption of the YPS genes coding for yapsin 1, 2, 3, 6, and 7 proteases in various combinations. The multiple YPS-deletion mutant strains did not show detectable growth defects under normal growth conditions, although some of them were hypersensitive to hygromycin B, acid (pH 3.5) and alkali (pH 8.0) conditions. The quintuple disruptant (yps1Deltayps2Deltayps3Deltayps6Deltayps7Delta) was the most efficient in preventing the proteolytic degradation of hPTH in fed-batch cultivations. The present data strongly indicate the involvement of other yapsin members besides Yps1p in the proteolysis of secretory recombinant proteins, particularly under high-density growth conditions.
Cho
Eun Young
EY
Korea Research Institute of Bioscience and Biotechnology, Daejeon, Republic of Korea.
Cheon
Seon Ah
SA
Kim
Hyunah
H
Choo
Jinho
J
Lee
Dong-Jik
DJ
Ryu
Ho Myung
HM
Rhee
Sang Ki
SK
Chung
Bong-Hyun
BH
Kim
Jeong-Yoon
JY
Kang
Hyun Ah
HA
D016282:Aspartic Acid Endopeptidases / Q000172:deficiency / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D010281:Parathyroid Hormone / Q000235:genetics / Q000378:metabolism; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism*; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000378:metabolism*; D029701:Saccharomyces cerevisiae Proteins / Q000235:genetics / Q000378:metabolism
C470125:PTH protein, human; D010281:Parathyroid Hormone; D011994:Recombinant Proteins; D029701:Saccharomyces cerevisiae Proteins; D016282:Aspartic Acid Endopeptidases; C119545:MKC7 protein, S cerevisiae; C119515:YPS3 protein, S cerevisiae; C499266:YPS1 protein, S cerevisiae