Recombinant human 92-kDa type IV collagenase/gelatinase from baculovirus-infected insect cells: expression, purification, and characterization.
Human 92-kDa type IV collagenase/gelatinase (MMP9) has been expressed in insect cells and secreted into the cell medium via a baculovirus expression system. The expression level of the proenzyme from Trichoplusia ni cells was estimated to be > = 300 mg/L of cell medium. The recombinant protein was purified in a single step using heparin-affinity chromatography with an overall yield of ca. 70%. The purified zymogen could be activated in vitro using 4-aminophenylmercuric acetate to yield an active protease. Kinetic analysis of the activated recombinant enzyme demonstrates that this material is comparable to activated MMP9 from natural human sources. The recombinant enzyme provides a useful source of protein for a variety of biochemical and biophysical studies aimed at elucidating the structure and function of human MMP9.
George
H J
HJ
DuPont Merck Research Laboratories, Wilmington, Delaware 19880-0400, USA.
Marchand
P
P
Murphy
K
K
Wiswall
B H
BH
Dowling
R
R
Giannaras
J
J
Hollis
G F
GF
Trzaskos
J M
JM
Copeland
R A
RA
D000595:Amino Acid Sequence; D000818:Animals; D002846:Chromatography, Affinity; D003001:Cloning, Molecular; D017364:Collagenases / Q000096:biosynthesis / Q000235:genetics* / Q000302:isolation & purification; D004789:Enzyme Activation / Q000187:drug effects; D004792:Enzyme Precursors / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D005822:Genetic Vectors; D006801:Humans; D007700:Kinetics; D020780:Matrix Metalloproteinase 9; D003012:Microbial Collagenase / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D008969:Molecular Sequence Data; D009036:Moths / Q000166:cytology / Q000378:metabolism; D017924:Nucleopolyhedroviruses / Q000235:genetics; D010455:Peptides / Q000378:metabolism; D010662:Phenylmercuric Acetate / Q000031:analogs & derivatives / Q000494:pharmacology; D011993:Recombinant Fusion Proteins / Q000302:isolation & purification / Q000378:metabolism; D013045:Species Specificity; D018411:Spodoptera / Q000166:cytology / Q000378:metabolism
D004792:Enzyme Precursors; D010455:Peptides; D011993:Recombinant Fusion Proteins; C010024:4-aminophenylmercuriacetate; D017364:Collagenases; D003012:Microbial Collagenase; D020780:Matrix Metalloproteinase 9; D010662:Phenylmercuric Acetate