Cloning, functional expression, and characterization of cystatin in sesame seed.
A cDNA fragment encoding cystatin, a cysteine protease inhibitor, was obtained from maturing sesame seeds. The clone was constructed in a nonfusion or fusion vector and then overexpressed in Escherichia coli. The recombinant cystatins were found in the soluble fraction of cell extract and were demonstrated to be functionally active in a reverse zymographic assay. The corresponding endogenous 22 kDa cystatin of low abundance in mature seeds was purified to homogeneity via a papain-coupling affinity column and confirmed by western blotting with antibodies against the recombinant cystatin. Both endogenous and recombinant cystatin proteins showed effective inhibitory activities against papain with K(i) values of 7.89 x 10(-8) M and 2.77 x 10(-8) M, respectively. Immunodetection indicated that cystatin was specifically expressed in maturing seeds and rapidly degraded in germination. Accordingly, zymographic and inhibition analyses showed that sesame cystatin could not inhibit the de novo synthesized proteases in germinating seeds. It is suggested that sesame cystatin may play a role in the regulation of endogenous cysteine proteases during seed maturation and germination.
Graduate Institute of Biotechnology, National Chung Hsing University, Taichung, Taiwan 40227, Republic of China.
Douglas J H
DJ
Shyu
Wing-Ming
WM
Chou
Tien-Joung
TJ
Yiu
Coney P C
CP
Lin
Jason T C
JT
Tzen
D000595:Amino Acid Sequence; D002352:Carrier Proteins / Q000037:antagonists & inhibitors; D003001:Cloning, Molecular; D015891:Cystatins / Q000737:chemistry / Q000235:genetics* / Q000494:pharmacology; D018076:DNA, Complementary / Q000302:isolation & purification; D018744:DNA, Plant / Q000737:chemistry / Q000302:isolation & purification; D004791:Enzyme Inhibitors / Q000494:pharmacology; D015870:Gene Expression; D008969:Molecular Sequence Data; D012639:Seeds / Q000737:chemistry*; D016415:Sequence Alignment; D031685:Sesamum / Q000737:chemistry*
D002352:Carrier Proteins; D015891:Cystatins; D018076:DNA, Complementary; D018744:DNA, Plant; D004791:Enzyme Inhibitors