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 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.
Hodges
D J
DJ
Rheumatology Research Unit, Addenbrookes Hospital, Cambridge, UK.
Reid
D G
DG
Rowan
A D
AD
Clark
I M
IM
Cawston
T E
TE
D000817:Anilino Naphthalenesulfonates; D003001:Cloning, Molecular; D017364:Collagenases / Q000378:metabolism; D005456:Fluorescent Dyes; D006801:Humans; D009682:Magnetic Resonance Spectroscopy; D017433:Protein Structure, Secondary; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics; D013113:Spin Labels; D013499:Surface Properties; D019715:Tissue Inhibitor of Metalloproteinase-1 / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism
D000817:Anilino Naphthalenesulfonates; D005456:Fluorescent Dyes; D011994:Recombinant Proteins; D013113:Spin Labels; D019715:Tissue Inhibitor of Metalloproteinase-1; C027132:1-anilino-8-naphthalenesulfonate; D017364:Collagenases