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) fusion protein was ligated into a pET-23a(+) expression vector and then transformed into Escherichia coli AD494(DE3) expression host. After induction by isopropyl beta-D-thiogalactopyranoside, a high level of the soluble form of recombinant trx-cystatin was expressed in the cytoplasm of E. coli. The recombinant trx-cystatin could be purified by Ni(2+)-NTA agarose affinity chromatography. The molecular mass (M) of the recombinant trx-cystatin was approximately 28 kDa composed of recombinant thioredoxin (16 kDa) and recombinant mature carp ovarian cystatin (12 kDa). Both recombinant trx-fused and mature carp ovarian cystatins were stable at pH 6-11. No obvious decrease in activity was observed even after 5 min of incubation at 60 degrees C. They exhibited papain-like protease inhibition activity comparable to that of the mature carp ovarian cystatin, which could inhibit papain and mackerel cathepsins L and L-like, but not cathepsin B.
Tzeng
S S
SS
Department of Food Science, National Taiwan Ocean University, Keelung, Taiwan 202, Republic of China.
Chen
G H
GH
Chung
Y C
YC
Jiang
S T
ST
D000818:Animals; D002347:Carps / Q000378:metabolism*; D015891:Cystatins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D015853:Cysteine Proteinase Inhibitors / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D004926:Escherichia coli / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D005260:Female; D006863:Hydrogen-Ion Concentration; D008970:Molecular Weight; D010053:Ovary
D015891:Cystatins; D015853:Cysteine Proteinase Inhibitors