中华鲟半胱氨酸蛋白酶抑制剂在毕赤酵母中的表达和活性分析

为研究鱼类半胱氨酸蛋白酶抑制剂（Cystatin）的功能并探索其在水产加工和病害防治中的应用潜力，将PCR改造后的编码成熟肽中华鲟（Acipenser sinensis）cystatin 基因亚克隆到毕赤酵母整合型表达载体pPICZαA，氯化锂法转化毕赤酵母菌株GS115，构建表达cystatin的酵母基因工程菌。经甲醇诱导、SDSPAGE检测培养基上清液，表明中华鲟cystatin在毕赤酵母中实现了高效表达，重组cystatin表达量约为215mg·L^-1。纯化后重组蛋白纯度达94.2%。生物活性检测结果表明，1μg重组中华鲟cystatin约能抑制15μg木瓜蛋白酶的水解活性。

Expression of Chinese Sturgeon Cystatin in Yeast Pichia pastoris and Its Proteinase Inhibitory Activity Analysis

Cystatin, which widely distributed in both tissues and body fluids of animal and plant, was a superfamily of cysteine proteinase inhibitors. It could form activity-inhibitor complexes with cysteine proteinases to inhibit the hydrolytic activity of proteinases. Cystatin played important roles not only in the inhibition of the proteolytic degradation of fish muscle, but also in biological defense systems against invaders. To explore the functions of fish cystatin and the potential values in fish disease prevention and cure, as well as seafood processing, the recombinant yeast strains which could express Chinese sturgeon cystatin were constructed. First, the cystatin cDNA of Chinese sturgeon, which had been PCR modified, was subcloned into yeast integrated vector pPICZaA. After extracted and purified, the recombinant plasmids were linearized by Sac I. The yeast Pichia pastoris GS115 strain was transformed by use of the Lithium Chloride transformation method, and the recombinant cystatin yeast strains got. After 0.5% methanol induction, SDS-PAGE analysis of the culture supernatant indicated that the yield of recombinant cystatin was about 215mg x L(-1) with the percentage about 73.6%. The recombinant cystatin was purified through Q-Sepharose anion-exchange chromatography, and the purity reached about 94.2%. The inhibitory activity of recombinant cystatin was measured by inhibiting the proteinase activity of papain. The results showed that about 1 microg recombinant cystatin could inhibit the activity of 15 microg papain. Heat stability assay results showed that there was a decrease in inhibitory activity of cystatin with the increasing of temperature. When solution of recombinant cystatin was kept at 70 degrees C for 5min, the inhibitory activity reduced fast. While the recombinant cystatin was heated to 90 degrees C for 5min, the inhibitory activity of recombinant cystatin was undetected. The inhibitory activity for recombinant Chinese sturgeon cystatin was higher than that of CPI (cysteine proteinase inhibitor) from seeds of corn, that about 1 microg purified CIP could inhibited the activity of 0.278 microg papain. But the heat stability of recombinant cystatin is lower than that of the corn CPI. The expression level and the activity of recombinant cystatin from yeast Pichia pastoris were higher than those from E. coli. Moreover, recombinant cystatin from Pichia pastoris was easier to separate and purify. This paper reported that recombinant fish cystatin was produced in a highly efficient expression system based on the methylotrophic yeast, further work will focus on the function of recombinant Chinese sturgeon cystatin to resist fish disease and explore the value of cystatin as a food additive to inhibit cysteine proteinases during surimi processing.