Molecular cloning, expression, and functional characterization of a cystatin from pineapple stem.
A cDNA fragment encoding the cysteine protease inhibitor, cystatin, was cloned from pineapple (Ananas comosus) stem. This clone was constructed in a fusion vector and was easily over-expressed in Escherichia coli; satisfactory over-expression of non-fusion cystatin was achieved after an additional start codon was inserted prior to its coding sequence. Both recombinant cystatins were predominately found in the soluble fraction of the cell extract, and were demonstrated to be functionally active in a reverse zymographic assay. The fusion and non-fusion cystatins were separately purified to homogeneity via a His-tag or papain-coupling affinity column. Effective inhibitory activity against papain was detected with both the fusion and non-fusion cystatins with comparable K(i) values of 1.18 x 10(-10) M and 9.53 x 10(-11) M, respectively. The recombinant cystatins were found to be thermally stable up to 60 degrees C. Inhibition of the endogenous protease activity in minced fish muscle revealed that the recombinant pineapple cystatins might be an adequate stabilizer to prevent protein degradation during industrial food processing.
Graduate Institute of Biotechnology, National Chung Hsing University, Taichung 40227, Taiwan.
Douglas J H
DJ
Shyu
Chia-Lin
CL
Chyan
Jason T C
JT
Tzen
Wing-Ming
WM
Chou
D000595:Amino Acid Sequence; D031230:Ananas / Q000378:metabolism*; D002846:Chromatography, Affinity; D003001:Cloning, Molecular; D018387:Codon, Initiator / Q000235:genetics*; D015891:Cystatins / Q000235:genetics* / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D008969:Molecular Sequence Data; D010206:Papain / Q000378:metabolism*; D011994:Recombinant Proteins / Q000235:genetics* / Q000378:metabolism
D018387:Codon, Initiator; D015891:Cystatins; D011994:Recombinant Proteins; D010206:Papain