Overexpression of the soluble form of chicken cystatin in Escherichia coli and its purification.
A cDNA encoding chicken cystatin was cloned into the pET-23a(+) expression vector and then transformed into Escherichia coli AD494(DE3)pLysS expression host. An active soluble form of cystatin was expressed in the cytoplasm of E. coli induced by isopropyl beta-D-thiogalactopyranoside. The recombinant chicken cystatin was purified to electrophoretic homogeneity by a simple and rapid method involving heat treatment and Sephacryl S-100 gel filtration chromatography. The recombinant cystatin behaved as a thermal-stable protein and exhibited papain-like protease inhibition activity comparable to the natural chicken cystatin.
Chen
G H
GH
Department of Food Science, National Taiwan Ocean University, Keelung.
Tang
S J
SJ
Chen
C S
CS
Jiang
S T
ST
D000818:Animals; D002403:Cathepsins / Q000037:antagonists & inhibitors; D002645:Chickens; D003001:Cloning, Molecular / Q000379:methods; D015891:Cystatins / Q000235:genetics* / Q000302:isolation & purification* / Q000494:pharmacology; D015853:Cysteine Proteinase Inhibitors / Q000302:isolation & purification* / Q000494:pharmacology; D018076:DNA, Complementary; D004926:Escherichia coli; D007700:Kinetics; D008168:Lung / Q000378:metabolism; D010206:Papain / Q000037:antagonists & inhibitors; D012333:RNA, Messenger / Q000235:genetics; D011994:Recombinant Proteins / Q000302:isolation & purification / Q000494:pharmacology
D015891:Cystatins; D015853:Cysteine Proteinase Inhibitors; D018076:DNA, Complementary; D012333:RNA, Messenger; D011994:Recombinant Proteins; D002403:Cathepsins; D010206:Papain