Cloning, functional expression and characterization of a phytocystatin gene from jelly fig (Ficus awkeotsang Makino) achenes

A cDNA clone encoding a phytocystatin was isolated and identified from about 300 expressed sequence tag (EST) clones in maturing jelly fig (Ficus awkeotsang Makino) achenes. This clone, named FaCYS, consists of 582 bp encoding 114 amino acids with a putative signal peptide. The predicted mature protein contains no cysteine and has a molecular mass of 10.8 kDa with an isoelectric point (pI) of 9.7. FaCYS constructed in nonfusion and fusion vectors were overexpressed in Escherichia coli as nonfusion and his-tagged recombinants, respectively. Both recombinants were found in the soluble fractions of the cell extracts. The purified nonfusion and his-tagged FaCYS exhibited papain inhibitory activity with similar Ki values of 2.7 x 10(-7) M and 2.4 x 10(-7) M, respectively. In addition, his-tagged recombinant proteins showed inhibitory activity toward human cathepsin B, cathepsin L and ficin with Ki values of 5.6 x 10(-7) M, 3.0 x 10(-8) M and 2.0 x 10(-7) M, but no inhibitory activity against stem bromelain. It was tolerant at a wide range of pH values and thermally stable up to 50 degrees C for 30 min. Furthermore, his-tagged FaCYS could arrest the fungal growth of Glomerella cingulata and Sclerotium rofsii.