title: Preparation of active recombinant TIMP-1 from Escherichia coli inclusion bodies and complex formation with the recombinant catalytic domain of PMNL-collagenase.
abstract: TIMP-1 is a member of the family of tissue inhibitors of metalloproteinases involved in regulating the activity of extracellular matrix degrading metalloproteinases. The TIMP-1 cDNA was obtained by reverse transcription-polymerase chain reaction (RT-PCR) amplification of the corresponding mRNA from human fibroblasts. Cloning and expression of the TIMP-1 cDNA were performed in Escherichia coli. In the host vector system chosen, rTIMP-1 is stored intracellularly in its denatured, insoluble form in inclusion bodies. We report a new method for the purification and renaturation of rTIMP-1 from E. coli inclusion bodies to an active inhibitor of matrix metalloproteinases (80% yield), presumably containing the correct assignment of the six disulfide bonds. A resin with the covalently bound recombinant catalytic domain of the PMNL-collagenase as the affinity ligand provided an effective means for the separation of correctly folded, active rTIMP-1 from inactive forms with mismatched disulfides. TIMP-1 and TIMP-2, the two most extensively examined members of the family of tissue inhibitors of metalloproteinases, are known to form a complex with the activated forms of most matrix metalloproteinases and the latent forms of the 92-kDa and 72-kDa gelatinases, respectively. In this study, we report on the complex formation of the recombinant catalytic domain of the PMNL-collagenase with TIMP-1, nonglycosylated recombinant TIMP-1, and recombinant TIMP-2. The Ki values for the different inhibitors were determined in a kinetic assay using a fluorogenic substrate peptide. In this assay, rTIMP-2 had a more effective inhibitory capability against the recombinant catalytic domain of the PMNL-collagenase than TIMP-1.(ABSTRACT TRUNCATED AT 250 WORDS)
authors.affiliation: Department of Biochemistry, Faculty of Chemistry, University of Bielefeld, Germany.
authors.forename: T
authors.initials: T
authors.lastname: Kleine
authors.forename: S
authors.initials: S
authors.lastname: Bartsch
authors.forename: J
authors.initials: J
authors.lastname: Bläser
authors.forename: S
authors.initials: S
authors.lastname: Schnierer
authors.forename: S
authors.initials: S
authors.lastname: Triebel
authors.forename: M
authors.initials: M
authors.lastname: Valentin
authors.forename: T
authors.initials: T
authors.lastname: Gote
authors.forename: H
authors.initials: H
authors.lastname: Tschesche
mesh_terms: D000595:Amino Acid Sequence; D001483:Base Sequence; D015153:Blotting, Western; D003001:Cloning, Molecular; D017364:Collagenases / Q000737:chemistry / Q000378:metabolism*; D017931:DNA Primers / Q000737:chemistry; D004926:Escherichia coli / Q000737:chemistry; D006023:Glycoproteins / Q000737:chemistry / Q000302:isolation & purification*; D006801:Humans; D007700:Kinetics; D046911:Macromolecular Substances; D020784:Matrix Metalloproteinase 8; D061965:Matrix Metalloproteinase Inhibitors; D008969:Molecular Sequence Data; D009363:Neoplasm Proteins / Q000737:chemistry; D009504:Neutrophils / Q000201:enzymology; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000302:isolation & purification*; D013329:Structure-Activity Relationship; D019716:Tissue Inhibitor of Metalloproteinase-2; D019714:Tissue Inhibitor of Metalloproteinases
chemical_list: D017931:DNA Primers; D006023:Glycoproteins; D046911:Macromolecular Substances; D061965:Matrix Metalloproteinase Inhibitors; D009363:Neoplasm Proteins; D011994:Recombinant Proteins; D019714:Tissue Inhibitor of Metalloproteinases; D019716:Tissue Inhibitor of Metalloproteinase-2; D017364:Collagenases; D020784:Matrix Metalloproteinase 8