Construction, expression, and characterization of a baculovirally expressed catalytic domain of human matrix metalloproteinase-9.
We report DNA construction, baculovirus expression, and partial characterization of a minienzyme form of the human matrix metalloproteinase-9 (MMP-9). The MMP-9 minienzyme gene construct consisting of the pre, pro, and catalytic domains of the MMP-9 was introduced into Sf9 insect cells using a baculovirus expression system. The expression of the recombinant MMP-9 minienzyme was estimated to be approximately 0.8 mg/L of cell medium. The recombinant protein was purified using a single-step gelatin-Sepharose affinity column and yielded a highly stable and active minienzyme with gelatinolytic activity. Moreover, two interesting findings related to MMP-9 interactions with heparin and TIMP-1 resulted from our studies. First, the pro and catalytic domains of the human MMP-9 are not sufficient for heparin affinity. Second, in contrast to the prevailing consensus, TIMP-1 blockade of the enzymatic activity of MMP-9 does not require prior binding to the C-terminus of its MMP-9 protein substrate.
Sadatmansoori
S
S
Department of Biochemistry and Biophysics, Texas A&M University, College Station, TX 77843, USA.
MacDougall
J
J
Khademi
S
S
Cooke
L S
LS
Guarino
L
L
Meyer
E F
EF
Forough
R
R
D000818:Animals; D016367:Baculoviridae / Q000235:genetics* / Q000378:metabolism; D001665:Binding Sites; D020134:Catalytic Domain; D002460:Cell Line; D003001:Cloning, Molecular; D004789:Enzyme Activation; D004795:Enzyme Stability; D015870:Gene Expression; D006493:Heparin / Q000378:metabolism; D006801:Humans; D020780:Matrix Metalloproteinase 9 / Q000737:chemistry* / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D011480:Protease Inhibitors / Q000494:pharmacology; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D018411:Spodoptera / Q000235:genetics; D019715:Tissue Inhibitor of Metalloproteinase-1 / Q000494:pharmacology; D014162:Transfection
D011480:Protease Inhibitors; D011994:Recombinant Proteins; D019715:Tissue Inhibitor of Metalloproteinase-1; D006493:Heparin; D020780:Matrix Metalloproteinase 9