Folding and characterization of the amino-terminal domain of human tissue inhibitor of metalloproteinases-1 (TIMP-1) expressed at high yield in E. coli.
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%.
Huang
W
W
Department of Biochemistry and Molecular Biology, University of Miami, FL 33134, USA.
Suzuki
K
K
Nagase
H
H
Arumugam
S
S
Van Doren
S R
SR
Brew
K
K
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
D006023:Glycoproteins; D011993:Recombinant Fusion Proteins; D019714:Tissue Inhibitor of Metalloproteinases