title: High-level expression and characterization of carboxypeptidase Y from Saccharomyces cerevisiae in Pichia pastoris GS115.
abstract: Carboxypeptidase Y is widely used in peptide sequencing and mass spectrometry. PRC1 coding for proteinase C from Saccharomyces cerevisiae was expressed in Pichia pastoris GS115 as procarboxypeptidase Y with a yield of ~605 mg/l in shake-flasks after 168 h induction with 1 % (v/v) methanol. This precursor of carboxypeptidase Y was cleaved by endogenous proteinases of P. pastoris and released into the fermentation broth as active carboxypeptidase Y within 2 weeks at 10 °C, which facilitated the preparation of mature carboxypeptidase Y. The recombinant enzyme was purified. It was optimally active at 30 °C and pH 6.0, with an optimal activity of ~305 U/mg using benzyloxycarbonyl-L-phenylalanyl-L-leucine as substrate. This is the first report about high-level expression and activation of carboxypeptidase Y in P. pastoris.
authors.affiliation: Hubei Collaborative Innovation Center for Green Transformation of Bio-resources, Hubei Key Laboratory of Industrial Biotechnology, College of Life Sciences, Hubei University, Wuhan, 430062, People's Republic of China.
authors.forename: Xianhong
authors.initials: X
authors.lastname: Yu
authors.forename: Chao
authors.initials: C
authors.lastname: Zhai
authors.forename: Xing
authors.initials: X
authors.lastname: Zhong
authors.forename: Wei
authors.initials: W
authors.lastname: Tang
authors.forename: Xiaojuan
authors.initials: X
authors.lastname: Wang
authors.forename: Hu
authors.initials: H
authors.lastname: Yang
authors.forename: Wanping
authors.initials: W
authors.lastname: Chen
authors.forename: Lixin
authors.initials: L
authors.lastname: Ma
mesh_terms: D043402:Cathepsin A / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D004795:Enzyme Stability; D010843:Pichia / Q000235:genetics / Q000378:metabolism*; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D012441:Saccharomyces cerevisiae / Q000201:enzymology / Q000235:genetics*
chemical_list: D011994:Recombinant Proteins; D043402:Cathepsin A