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, w as expressed in the methylotrophic yeast Pichia pastoris by placing th e CTSE cDNA under the control of the methanol inducible alcohol oxidas e promoter. The human CTSE expressed in P. pastoris was secreted into the culture medium as an active enzyme directed by its native signal s equence despite its intracellular localization in mammalian cells. The time course analysis of the culture supernatant of the P. pastoris tr ansformant expressing human CTSE revealed that the recombinant human C TSE 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 t he transformant was performed in a high cell density fermenter and the recombinant human CTSE was highly purified from the culture supernata nt. The purified recombinant cathepsin E had the molecular mass of 82 kDa with the amino-terminal sequence starting with Ile(3.7) of the seq uence deduced from its cDNA sequence, suggesting that the human cathep sin E was accumulated in the culture supernatant as mature dimeric enz yme. The result of endoglycosidase-H digestion followed by Western blo t analysis of the purified recombinant cathepsin E suggested that the human cathepsin E expressed in P. pastoris received N-linked high-mann ose type glycosylation. The enzymatic properties of the recombinant en zyme were comparable to those of natural human CTSE.