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cDNA cloning, identification and characterization of a novel cystatin from the tentacle of Cyanea capillata
- Source :
-
Biochimie . Oct2003, Vol. 85 Issue 10, p1033. 7p. - Publication Year :
- 2003
-
Abstract
- 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, Ser97-Trp98, 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 Mr = 12,800 displayed an obvious inhibition of papain (Ki 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 °C, but unstable at neutral pH at >50 °C. [Copyright &y& Elsevier]
- Subjects :
- *BIOTECHNOLOGY
*ESCHERICHIA coli
*CHROMATOGRAPHIC analysis
*THIOREDOXIN
Subjects
Details
- Language :
- English
- ISSN :
- 03009084
- Volume :
- 85
- Issue :
- 10
- Database :
- Academic Search Index
- Journal :
- Biochimie
- Publication Type :
- Academic Journal
- Accession number :
- 11537368
- Full Text :
- https://doi.org/10.1016/S0300-9084(03)00132-9