Production and purification of human fibroblast collagenase (MMP-1) expressed in the methylotrophic yeast Pichia pastoris

• Published in: Protein expression and purification Vol. 7; no. 4; pp. 423 - 430
• Main Authors: Rosenfeld, S A, Ross, O H, Hillman, M C, Corman, J I, Dowling, R L
• Format: Journal Article
• Language: English
• Published: United States 01.06.1996
• Subjects: Amino Acid Sequence; Collagenases - biosynthesis; Collagenases - chemistry; Collagenases - genetics; Collagenases - isolation & purification; DNA, Fungal - genetics; Electrophoresis, Polyacrylamide Gel; Enzyme Activation; Fibroblasts - enzymology; Fungal Proteins - biosynthesis; Fungal Proteins - chemistry; Fungal Proteins - genetics; Fungal Proteins - isolation & purification; Gene Expression; Genetic Vectors; Humans; Matrix Metalloproteinase 1; Molecular Sequence Data; Phenylmercuric Acetate - analogs & derivatives; Phenylmercuric Acetate - chemistry; Pichia - enzymology; Pichia - genetics; Recombinant Proteins - chemistry; Recombinant Proteins - genetics; Recombinant Proteins - isolation & purification; Recombinant Proteins - metabolism; Sequence Analysis; Sodium Dodecyl Sulfate - chemistry
• ISSN: 1046-5928
• DOI: 10.1006/prep.1996.0063

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.