title: Production and purification of human fibroblast collagenase (MMP-1) expressed in the methylotrophic yeast Pichia pastoris.
abstract: The cDNA that encodes the proenzyme form of human fibroblast collagenase (proMMP-1) was expressed in the methylotrophic yeast Pichia pastoris. 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+ Muts (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 fermentations. The protein was purified to greater than 95% homogeneity. The recombinant proMMP-1 was comparable to the native fibroblast material based on (i) migration of the full-length molecule as a 52-kDa protein on reducing SDS-PAGE, (ii) correct N-terminal amino acid sequence, (iii) activation of the full-length molecule by 4-amino-phenylmercuric acetate to yield processed protein species, (iv) degradation of gelatin as monitored by zymogram gels, and (v) enzymatic activity. These data suggest that the P. pastoris expression system offers a convenient and efficient means to produce and purify MMP-1.
authors.affiliation: Dupont-Merck Pharmaceutical Company, Wilmington, Delaware 19880, USA.
authors.forename: S A
authors.initials: SA
authors.lastname: Rosenfeld
authors.forename: O H
authors.initials: OH
authors.lastname: Ross
authors.forename: M C
authors.initials: MC
authors.lastname: Hillman
authors.forename: J I
authors.initials: JI
authors.lastname: Corman
authors.forename: R L
authors.initials: RL
authors.lastname: Dowling
mesh_terms: D000595:Amino Acid Sequence; D017364:Collagenases / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification*; D004271:DNA, Fungal / Q000235:genetics; D004591:Electrophoresis, Polyacrylamide Gel; D004789:Enzyme Activation; D005347:Fibroblasts / Q000201:enzymology*; D005656:Fungal Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification; D015870:Gene Expression; D005822:Genetic Vectors; D006801:Humans; D020781:Matrix Metalloproteinase 1; D008969:Molecular Sequence Data; D010662:Phenylmercuric Acetate / Q000031:analogs & derivatives / Q000737:chemistry; D010843:Pichia / Q000201:enzymology / Q000235:genetics; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D017421:Sequence Analysis; D012967:Sodium Dodecyl Sulfate / Q000737:chemistry
chemical_list: D004271:DNA, Fungal; D005656:Fungal Proteins; D011994:Recombinant Proteins; D012967:Sodium Dodecyl Sulfate; D017364:Collagenases; D020781:Matrix Metalloproteinase 1; D010662:Phenylmercuric Acetate