Secretion of human intracellular aspartic proteinase cathepsin E expressed in the methylotrophic yeast, Pichia pastoris and characterization of produced recombinant cathepsin E.
The human gastric cathepsin E (CTSE), a dimeric aspartic proteinase, was expressed in the methylotrophic yeast Pichia pastoris by placing the CTSE cDNA under the control of the methanol inducible alcohol oxidase promoter. The human CTSE expressed in P. pastoris was secreted into the culture medium as an active enzyme directed by its native signal sequence despite its intracellular localization in mammalian cells. The time course analysis of the culture supernatant of the P. pastoris transformant expressing human CTSE revealed that the recombinant human CTSE was secreted as a 90 kDa molecule and then converted via an 84 kDa intermediate to an 82 kDa mature molecule. A large-scale culture of the transformant was performed in a high cell density fermentor and the recombinant human CTSE was highly purified from the culture supernatant. The purified recombinant cathepsin E had the molecular mass of 82 kDa with the amino-terminal sequence starting with Ile37 of the sequence deduced from its cDNA sequence, suggesting that the human cathepsin E was accumulated in the culture supernatant as mature dimeric enzyme. The result of endoglycosidase-H digestion followed by Western blot analysis of the purified recombinant cathepsin E suggested that the human cathepsin E expressed in P. pastoris received N-linked high-mannose type glycosylation. The enzymatic properties of the recombinant enzyme were comparable to those of natural human CTSE.
Yamada
M
M
Biotechnology Research Laboratory, Tosoh Corporation, Kanagawa, Japan.
Azuma
T
T
Matsuba
T
T
Iida
H
H
Suzuki
H
H
Yamamoto
K
K
Kohli
Y
Y
Hori
H
H
D001483:Base Sequence; D020059:Cathepsin E; D002403:Cathepsins / Q000096:biosynthesis* / Q000235:genetics* / Q000378:metabolism; D006026:Glycoside Hydrolases; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008969:Molecular Sequence Data; D010843:Pichia / Q000737:chemistry / Q000201:enzymology*; D011994:Recombinant Proteins / Q000096:biosynthesis* / Q000737:chemistry; D013270:Stomach / Q000201:enzymology*
D011994:Recombinant Proteins; D006026:Glycoside Hydrolases; D002403:Cathepsins; D020059:Cathepsin E