Abstract:

Prokaryotic expression, purification and identification of a cysteine proteinase inhibitor, cystatin, from
Hypophthalmichthys molitrix were performed, and the antibacterial effect of the recombinant cystatin on Pseudomonas
aeruginosa was investigated in this work. The cystatin was expressed by E. coli BL21 (DE3) transformed with pET-30-
Cystatin with the induction of isopropyl-beta-D-galactose (IPTG), and purified by Ni2+-NTA affinity chromatography. The
purified protein appeared as a single band on SDS-PAGE, corresponding to a molecular weight of approximately 20.6 kD.
The same protein also appeared as a single active peak on the gel filtration HPLC of TSK-GEL G2000SW with a purity of
94.27% and a relative molecular weight of 20.9 kD, as calibrated by standards. The inhibitory activity of the cystatin against papain
(45.375 μmol) was 0.718 μg, which was determined by inhibitor titration method with Azocasein as the substrate. The antibacterial
effect of the recombinant cystatin on Pseudomonas aeruginosa was tested by filter paper diffusion method, and it was found
that the diameters of the inhibition zones were 8, 9, 13 and 26 mm with the addition of 20, 60, 120 and 200 units of the cystatin,
respectively. This suggests that the antibacterial activity of the cystatin on Pseudomonas aeruginosa is dose-dependent.

Key words: cystatin, Hypophthalmichthys molitrix, prokaryotic expression, identification, Pseudomonas aeruginosa;antibacterial activity