Preparation of recombinant tissue inhibitor of metalloproteinases-1 (TIMP-1) in high yield and identification of a hydrophobic surface feature

Eur J Biochem. 1998 Nov 1;257(3):562-9. doi: 10.1046/j.1432-1327.1998.2570562.x.


The work presented here describes an effective method for refolding recombinant tissue inhibitor of metalloproteinases-1 (TIMP-1), a 21-kDa protein with six disulphide bonds. A yield of 30 mg TIMP-1/l culture medium was obtained from a high level bacterial expression system, using a slow removal of denaturant in the presence of 0.5 M guanidine and a suitable redox buffer. This protein is identical to the wild-type species when specific activity and secondary structure (by CD) are compared. The fluorescent, hydrophobic compound 8-anilino 1-naphthalene sulphonate (ANS) was used to quantify hydrophobic binding sites on the surface of both wild-type and recombinant TIMP-1. The wild-type protein has 1 binding site with a mean Kd of 1.3 mM and the recombinant protein has 1.5 binding sites with a mean Kd of 0.39 mM. The presence of surface hydrophobic residues is confirmed by selective broadening of ethyl and aromatic signals in the 1H-NMR spectrum on the addition of the paramagnetic probe 4-hydroxy-2,2,6,6-tetramethylpiperidinyl-N-oxy, OH-TEMPO, to wild-type TIMP-1. When wild-type TIMP-1 is incubated with the N-terminal fragment of human fibroblast collagenase prior to the addition of ANS, the number of binding sites in the system decreases to 0.5 with a Kd of 0.15 mM.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Anilino Naphthalenesulfonates
  • Cloning, Molecular
  • Collagenases / metabolism
  • Fluorescent Dyes
  • Humans
  • Magnetic Resonance Spectroscopy
  • Protein Structure, Secondary
  • Recombinant Proteins / chemistry
  • Recombinant Proteins / genetics
  • Spin Labels
  • Surface Properties
  • Tissue Inhibitor of Metalloproteinase-1 / chemistry
  • Tissue Inhibitor of Metalloproteinase-1 / genetics*
  • Tissue Inhibitor of Metalloproteinase-1 / metabolism


  • Anilino Naphthalenesulfonates
  • Fluorescent Dyes
  • Recombinant Proteins
  • Spin Labels
  • Tissue Inhibitor of Metalloproteinase-1
  • 1-anilino-8-naphthalenesulfonate
  • Collagenases