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Folding and characterization of the amino‐terminal domain of human tissue inhibitor of metalloproteinases‐1 (TIMP‐1) expressed at high yield in E. coli
Author(s) -
Huang Wen,
Suzuki Ko,
Nagase Hideaki,
Arumugan S.,
Van Doren Steven R.,
Brew Keith
Publication year - 1996
Publication title -
febs letters
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.593
H-Index - 257
eISSN - 1873-3468
pISSN - 0014-5793
DOI - 10.1016/0014-5793(96)00304-3
Subject(s) - matrix metalloproteinase , recombinant dna , chemistry , yield (engineering) , escherichia coli , proteolysis , protein folding , folding (dsp implementation) , biochemistry , nuclear magnetic resonance spectroscopy , inclusion bodies , biophysics , microbiology and biotechnology , enzyme , stereochemistry , biology , materials science , gene , electrical engineering , metallurgy , engineering
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 15 N‐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%.