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 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.
Rosenfeld
S A
SA
Dupont-Merck Pharmaceutical Company, Wilmington, Delaware 19880, USA.
Ross
O H
OH
Hillman
M C
MC
Corman
J I
JI
Dowling
R L
RL
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
D004271:DNA, Fungal; D005656:Fungal Proteins; D011994:Recombinant Proteins; D012967:Sodium Dodecyl Sulfate; D017364:Collagenases; D020781:Matrix Metalloproteinase 1; D010662:Phenylmercuric Acetate