The expression, refolding, and purification of the catalytic domain of human collagenase-3 (MMP-13)

被引：91

作者：

Pathak, N ^{[1
]}

Hu, SI ^{[1
]}

Koehn, JA ^{[1
]}

机构：

[1] Novartis Inst Biomed Res, Summit, NJ 07901 USA

来源：

PROTEIN EXPRESSION AND PURIFICATION | 1998年 / 14卷 / 02期

关键词：

D O I：

10.1006/prep.1998.0972

中图分类号：

Q5 [生物化学];

学科分类号：

071010 ; 081704 ;

摘要：

We have cloned, expressed, and purified a recombinant C-terminal truncated form (residues Glu103-Asn274) of human collagenase-3 (MMP-13) in Escherichia coli. The molecule contains the catalytic domain of the enzyme and is expressed almost exclusively as inclusion bodies. Using a combination of rapid dilution and diafiltration, the enzyme has been successfully refolded from these inclusion bodies. The protein was purified to homogeneity using cation-exchange and size exclusion chromatography. The purified enzyme is a monomer with a M-r of similar to 19,600 and was characterized using a variety of techniques including, SDS-PAGE, RP-HPLC, LC-MS, amino acid analysis, and dynamic light scattering. Microheterogeneity at the NH2-terminus of the refolded, purified protein disappeared after incubating for 30-60 min at 37 degrees C. The enzyme was highly active using a fluorescent peptide substrate and was found to release S-GAC: from bovine nasal cartilage chips. (C) 1998 Academic Press.

引用

页码：283 / 288

页数：6

共 22 条

[1] STROMELYSIN-1 - 3-DIMENSIONAL STRUCTURE OF THE INHIBITED CATALYTIC DOMAIN AND OF THE C-TRUNCATED PROENZYME [J].

BECKER, JW ;

MARCY, AI ;

ROKOSZ, LL ;

AXEL, MG ;

BURBAUM, JJ ;

FITZGERALD, PMD ;

CAMERON, PM ;

ESSER, CK ;

HAGMANN, WK ;

HERMES, JD ;

SPRINGER, JP .

PROTEIN SCIENCE, 1995, 4 (10) :1966-1976

[2] THE X-RAY CRYSTAL-STRUCTURE OF THE CATALYTIC DOMAIN OF HUMAN NEUTROPHIL COLLAGENASE INHIBITED BY A SUBSTRATE-ANALOG REVEALS THE ESSENTIALS FOR CATALYSIS AND SPECIFICITY [J].

BODE, W ;

REINEMER, P ;

HUBER, R ;

KLEINE, T ;

SCHNIERER, S ;

TSCHESCHE, H .

EMBO JOURNAL, 1994, 13 (06) :1263-1269

[3] STRUCTURE OF THE CATALYTIC DOMAIN OF HUMAN FIBROBLAST COLLAGENASE COMPLEXED WITH AN INHIBITOR [J].

BORKAKOTI, N ;

WINKLER, FK ;

WILLIAMS, DH ;

DARCY, A ;

BROADHURST, MJ ;

BROWN, PA ;

JOHNSON, WH ;

MURRAY, EJ .

NATURE STRUCTURAL BIOLOGY, 1994, 1 (02) :106-110

[4] Batimastat, a potent matrix metalloproteinase inhibitor, exhibits an unexpected mode of binding [J].

Botos, I ;

Scapozza, L ;

Zhang, DC ;

Liotta, LA ;

Meyer, EF .

PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA, 1996, 93 (07) :2749-2754

[5]

BRADFORD MM, 1976, ANAL BIOCHEM, V72, P248, DOI 10.1016/0003-2697(76)90527-3

[6] 1.8 angstrom structure of Hypoderma lineatum collagenase: A member of the serine proteinase family [J].

Broutin, I ;

Arnoux, B ;

Riche, C ;

Lecroisey, A ;

Keil, B ;

Pascard, C ;

Ducruix, A .

ACTA CRYSTALLOGRAPHICA SECTION D-STRUCTURAL BIOLOGY, 1996, 52 :380-392

[7]

FREIJE JMP, 1994, J BIOL CHEM, V269, P16766

[8]

Gooley PR, 1996, J BIOMOL NMR, V7, P8

[9] THE NMR STRUCTURE OF THE INHIBITED CATALYTIC DOMAIN OF HUMAN STROMELYSIN-1 [J].

GOOLEY, PR ;

OCONNELL, JF ;

MARCY, AI ;

CUCA, GC ;

SALOWE, SP ;

BUSH, BL ;

HERMES, JD ;

ESSER, CK ;

HAGMANN, WK ;

SPRINGER, JP ;

JOHNSON, BA .

NATURE STRUCTURAL BIOLOGY, 1994, 1 (02) :111-118

[10] SECONDARY STRUCTURE AND ZINC LIGATION OF HUMAN RECOMBINANT SHORT-FORM STROMELYSIN BY MULTIDIMENSIONAL HETERONUCLEAR NMR [J].

GOOLEY, PR ;

JOHNSON, BA ;

MARCY, AI ;

CUCA, GC ;

SALOWE, SP ;

HAGMANN, WK ;

ESSER, CK ;

SPRINGER, JP .

BIOCHEMISTRY, 1993, 32 (48) :13098-13108

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