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 expressi on system to produce glycosylation-free human procathepsin K and have devel oped conditions for refolding this enzyme. Recombinant human procathepsin K (EC 3.4.22.38) was expressed in E, coli, refolded from inclusion bodies, a nd further purified by Superdex 75 size-exclusion chromatography, Purified procathepsin K had a [MH](+) of 35,063 Da which is in agreement with the pr edicted mass of the construct, Amino-terminal sequence analysis matched the predicted sequence with no secondary sequence detected, Purified procathep sin K activated under autocatalytic conditions to a final specific activity of 23 mu mol 7-amido-4-methylcoumarin liberated/min/mg of enzyme using the fluorescent peptide substrate benzyloxycarbonyl-phenylalanine-arginine 7-a mido-4-methylcoumarin. This expression and refolding procedure yielded 50 m g of purified, glycosylation-free human procathepsin K from 1 liter of E, c oli cell culture and enabled the determination of the structure of human pr ocathepsin K at 2.6 Angstrom resolution. (C) 1999 Academic Press.