Expression of soluble form carp (Cyprinus carpio) ovarian cystatin in Escherichia coli and its purification

A DNA encoding thioredoxin- mature carp ovarian cystatin (trx-cystatin) fus ion protein was ligated into a pET-23a(+) expression vector and then transf ormed into Escherichia coli AD494(DE3) expression host. After induction by isopropyl beta -D-thiogalactopyranoside, a high level of the soluble form o f recombinant trx-cystatin was expressed in the cytoplasm of E. coli. The r ecombinant trx-cystatin could be purified by Ni2+-NTA agarose affinity chro matography. The molecular mass (M) of the recombinant trx-cystatin was simi lar to 28 kDa composed of recombinant thioredoxin (16 kDa) and recombinant mature carp ovarian cystatin (12 kDa). Both recombinant trx-fused and matur e carp ovarian cystatins were stable at pH 6-11. No obvious decrease in act ivity was observed even after 5 min of incubation at 60 degreesC. They exhi bited papain-like protease inhibition activity comparable to that of the ma ture carp ovarian cystatin, which could inhibit papain and mackerel catheps ins L and L-like, but not cathepsin B.