PRODUCTION AND PURIFICATION OF HUMAN FIBROBLAST COLLAGENASE (MMP-1) EXPRESSED IN THE METHYLOTROPHIC YEAST PICHIA-PASTORIS

The cDNA that encodes the proenzyme form of human fibroblast collagena se (proMMP-1) was expressed in the methylotrophic yeast Pichia pastori s. The proMMP-1 encoding DNA was fused to the Saccharomyces cerevisiae pre-pro alpha-mating factor secretion signal in the P. pastoris pPIC9 expression plasmid, transformed into strain GS115 (His(-)), and His() Mut(s) (slow methanol utilization) transformants were selected. Full -length proenzyme and processed forms of the protein could be detected in yeast culture supernatants following shake flask and 10-liter ferm entations. The protein was purified to greater than 95% homogeneity. T he recombinant proMMP-1 was comparable to the native fibroblast materi al based on (i) migration of the full-length molecule as a 52-kDa prot ein on reducing SDS-PAGE, (ii) correct N-terminal amino acid sequence, (iii) activation of the full-length molecule by 4-amino-phenylmercuri c acetate to yield processed protein species, (iv) degradation of gela tin as monitored by zymogram gels, and (v) enzymatic activity. These d ata suggest that the P. pastoris expression system offers a convenient and efficient means to produce and purify MMP-1. (C) 1996 Academic Pr ess, Inc.