PREPARATION OF RECOMBINANT TISSUE INHIBITOR OF METALLOPROTEINASES-1 (TIMP-1) IN HIGH-YIELD AND IDENTIFICATION OF A HYDROPHOBIC SURFACE-FEATURE

The work presented here describes an effective method for refolding re combinant tissue inhibitor of metalloproteinases-1 (TIMP-1), a 21-kDa protein with six disulphide bonds. A yield of 30 mg TIMP-1/l culture m edium was obtained from a high level bacterial expression system, usin g 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 sp ecies when specific activity and secondary structure (by CD) are compa red. The fluorescent, hydrophobic compound 8-anilino 1-naphthalene sul phonate (ANS) was used to quantify hydrophobic binding sites on the su rface of both wild-type and recombinant TIMP-1. The wild-type protein has 1 binding site with a mean K-d of 1.3 mM and the recombinant prote in has 1.5 binding sites with a mean K-d of 0.39 mM. The presence of s urface hydrophobic residues is confirmed by selective broadening of et hyl and aromatic signals in the H-1-NMR spectrum on the addition of th e paramagnetic probe 4-hydroxy-2,2,6,6-tetramethylpiperidinyl-N-oxy, O H-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 a ddition of ANS, the number of binding sites in the system decreases to 0.5 with a K-d of 0.15 mM.