Expression in Escherichia coli, refolding, and purification of human procathepsin K, an osteoclast-specific protease.

We have constructed and optimized a high yielding Escherichia coli expression system to produce glycosylation-free human procathepsin K and have developed conditions for refolding this enzyme. Recombinant human procathepsin K (EC 3.4.22.38) was expressed in E. coli, refolded from inclusion bodies, and further purified by Superdex 75 size-exclusion chromatography. Purified procathepsin K had a [MH]+ of 35,063 Da which is in agreement with the predicted mass of the construct. Amino-terminal sequence analysis matched the predicted sequence with no secondary sequence detected. Purified procathepsin K activated under autocatalytic conditions to a final specific activity of 23 micromol 7-amido-4-methylcoumarin liberated/min/mg of enzyme using the fluorescent peptide substrate benzyloxycarbonyl-phenylalanine-arginine-7-amido-4-methylcoumarin. This expression and refolding procedure yielded 50 mg of purified, glycosylation-free human procathepsin K from 1 liter of E. coli cell culture and enabled the determination of the structure of human procathepsin K at 2.6 A resolution.