Production, purification, and characterization of human matrilysin (PUMP) from recombinant Chinese hamster ovary cells.
A process for semicontinuous production of matrilysin zymogen secreted from recombinant Chinese hamster ovary (CHO) cells was developed. The zymogen was purified to apparent homogeneity by sequential ion-exchange and metal chelation chromatography. These processes were scaled-up to purify gram quantities of the zymogen. The N-terminus of the secreted zymogen from the recombinant cells was the same as the observed sequence of the zymogen from natural sources. Furthermore, activation and autocatalysis of the recombinant zymogen resulted in a form with an N-terminus identical to that of the corresponding native enzyme. The three C-terminal amino acids of both the recombinant zymogen and the corresponding smaller activated form were missing. Activated matrilysin was shown to have activity against a synthetic peptide substrate. The large quantities of matrilysin that can be produced and purified from the recombinant CHO cells will be useful in determination of the structure of matrilysin.
Barnett
J
J
Institute of Bio-organic Chemistry, Syntex Discovery Research, Palo Alto, California 94304.
Straub
K
K
Nguyen
B
B
Chow
J
J
Suttman
R
R
Thompson
K
K
Tsing
S
S
Benton
P
P
Schatzman
R
R
Chen
M
M
D000595:Amino Acid Sequence; D000818:Animals; D016466:CHO Cells; D002614:Chelating Agents; D002851:Chromatography, High Pressure Liquid; D002852:Chromatography, Ion Exchange; D006224:Cricetinae; D003412:Cricetulus; D004792:Enzyme Precursors / Q000096:biosynthesis* / Q000302:isolation & purification / Q000378:metabolism; D006801:Humans; D013058:Mass Spectrometry; D020783:Matrix Metalloproteinase 7; D008666:Metalloendopeptidases / Q000096:biosynthesis* / Q000302:isolation & purification / Q000378:metabolism; D008969:Molecular Sequence Data; D008970:Molecular Weight; D010446:Peptide Fragments / Q000138:chemical synthesis / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis* / Q000302:isolation & purification / Q000378:metabolism
D002614:Chelating Agents; D004792:Enzyme Precursors; D010446:Peptide Fragments; D011993:Recombinant Fusion Proteins; D008666:Metalloendopeptidases; D020783:Matrix Metalloproteinase 7