cDNA cloning, identification and characterization of a novel cystatin from the tentacle of Cyanea capillata.
Cystatin is of interest from biochemical and evolutionary prospective, and also has been applied in biotechnology. In this paper, a novel cystatin was found by EST sequence analysis of the cDNA library of Cyanea capillata tentacle. The sequence of a full-length cDNA clone contained an open reading frame encoding a putative 18-residue signal peptide and a mature protein of 113 amino acids, which showed only 26% identities to Family 2 cystatins and had its own characteristic enzyme-binding motifs, Ser(97)-Trp(98), which had not been found in any other known cystatins. Thus, the novel cystatin cloned from jellyfish was designated as cystatin J, which may belong to a new family of cystatin, called Family 4. The mature cystatin J was produced in Escherichia coli as a thioredoxin (Trx) fusion protein using the pET expression system and purified by affinity and cation exchange chromatography. The recombinant cystatin J of approximately M(r) = 12,800 displayed an obvious inhibition of papain (K(i) value below 0.5 nM), in competition with substrate. Thus, the recombinant cystatin J was a functional cystatin in spite of relatively lower sequence similarity with other cystatins. Activity of the novel cystatin was stable at pH 4-11 at 4 degrees C, but unstable at neutral pH at >50 degrees C.
Yang
Yanzhen
Y
Department of Biochemistry, Open Laboratory for Marine Functional Genomics of National High-Tech Development Program, College of Life Sciences, Sun Yat-Sen (Zhongshan) University, Guangzhou, PR China.
Cun
Shujian
S
Peng
Lisheng
L
Xie
Xiaojin
X
Wei
Jianwen
J
Yang
Wenli
W
Xu
Anlong
A
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D003001:Cloning, Molecular; D015891:Cystatins / Q000737:chemistry* / Q000145:classification / Q000235:genetics*; D018076:DNA, Complementary; D007700:Kinetics; D008969:Molecular Sequence Data; D010206:Papain / Q000037:antagonists & inhibitors / Q000737:chemistry; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D037401:Scyphozoa / Q000737:chemistry* / Q000235:genetics
D015891:Cystatins; D018076:DNA, Complementary; D011994:Recombinant Proteins; D010206:Papain