title: Folding and characterization of the amino-terminal domain of human tissue inhibitor of metalloproteinases-1 (TIMP-1) expressed at high yield in E. coli.
abstract: Methods are described for producing an active amino-terminal domain of tissue inhibitor of metalloproteinases-1 (N-TIMP-1) from inactive protein expressed as inclusion bodies in E. coli. Yields exceed 20 mg per litre of bacterial culture. Activity measurements, CD spectroscopy and NMR spectroscopy of the 15N-labeled protein show that it is fully active, homogeneous in conformation and suitable for high-resolution structural analysis. The affinity of N-TIMP-1 for matrix metalloproteinases 1, 2 and 3 is 6-8-fold less than that of the recombinant full-length protein, indicating that deletion of the C-terminal domain reduces the free energy of interaction by < 10%.
authors.affiliation: Department of Biochemistry and Molecular Biology, University of Miami, FL 33134, USA.
authors.forename: W
authors.initials: W
authors.lastname: Huang
authors.forename: K
authors.initials: K
authors.lastname: Suzuki
authors.forename: H
authors.initials: H
authors.lastname: Nagase
authors.forename: S
authors.initials: S
authors.lastname: Arumugam
authors.forename: S R
authors.initials: SR
authors.lastname: Van Doren
authors.forename: K
authors.initials: K
authors.lastname: Brew
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D016466:CHO Cells; D002942:Circular Dichroism; D006224:Cricetinae; D004926:Escherichia coli / Q000378:metabolism*; D006023:Glycoproteins / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics; D006801:Humans; D009682:Magnetic Resonance Spectroscopy; D008969:Molecular Sequence Data; D011401:Promoter Regions, Genetic; D011487:Protein Conformation; D017510:Protein Folding; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis* / Q000737:chemistry; D013816:Thermodynamics; D019714:Tissue Inhibitor of Metalloproteinases
chemical_list: D006023:Glycoproteins; D011993:Recombinant Fusion Proteins; D019714:Tissue Inhibitor of Metalloproteinases