title: Multiple-yapsin-deficient mutant strains for high-level production of intact recombinant proteins in Saccharomyces cerevisiae.
abstract: 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.
authors.affiliation: Korea Research Institute of Bioscience and Biotechnology, Daejeon, Republic of Korea.
authors.forename: Eun Young
authors.initials: EY
authors.lastname: Cho
authors.forename: Seon Ah
authors.initials: SA
authors.lastname: Cheon
authors.forename: Hyunah
authors.initials: H
authors.lastname: Kim
authors.forename: Jinho
authors.initials: J
authors.lastname: Choo
authors.forename: Dong-Jik
authors.initials: DJ
authors.lastname: Lee
authors.forename: Ho Myung
authors.initials: HM
authors.lastname: Ryu
authors.forename: Sang Ki
authors.initials: SK
authors.lastname: Rhee
authors.forename: Bong-Hyun
authors.initials: BH
authors.lastname: Chung
authors.forename: Jeong-Yoon
authors.initials: JY
authors.lastname: Kim
authors.forename: Hyun Ah
authors.initials: HA
authors.lastname: Kang
mesh_terms: 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
chemical_list: 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