Ma Dong-Mei, Bai Jun-Jie, Jian Qing, Lao Hai-Hua, Ye Xing, Luo Jian-Ren
Pearl River Fisheries Research Institute, Chinese Academy of Fishery Sciences, Guangzhou 510380, China.
Sheng Wu Gong Cheng Xue Bao. 2003 Sep;19(5):598-602.
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.
半胱氨酸蛋白酶抑制剂广泛分布于动植物的组织和体液中，是一个半胱氨酸蛋白酶抑制剂超家族。它能与半胱氨酸蛋白酶形成活性-抑制剂复合物，从而抑制蛋白酶的水解活性。半胱氨酸蛋白酶抑制剂不仅在抑制鱼肉的蛋白水解降解方面发挥重要作用，还在抵御外来侵害的生物防御系统中发挥作用。为了探索鱼类半胱氨酸蛋白酶抑制剂的功能及其在鱼类疾病防治和海产品加工中的潜在价值，构建了能够表达中华鲟半胱氨酸蛋白酶抑制剂的重组酵母菌株。首先，将经过PCR修饰的中华鲟半胱氨酸蛋白酶抑制剂cDNA亚克隆到酵母整合载体pPICZaA中。提取并纯化后，用Sac I将重组质粒线性化。采用氯化锂转化法转化酵母毕赤酵母GS115菌株，获得重组半胱氨酸蛋白酶抑制剂酵母菌株。经0.5%甲醇诱导后，对培养上清进行SDS-PAGE分析，结果表明重组半胱氨酸蛋白酶抑制剂的产量约为215mg·L⁻¹，占比约为73.6%。通过Q-Sepharose阴离子交换层析法纯化重组半胱氨酸蛋白酶抑制剂，纯度达到约94.2%。通过抑制木瓜蛋白酶的蛋白酶活性来测定重组半胱氨酸蛋白酶抑制剂的抑制活性。结果表明，约1μg重组半胱氨酸蛋白酶抑制剂可抑制15μg木瓜蛋白酶的活性。热稳定性测定结果表明，随着温度升高，半胱氨酸蛋白酶抑制剂的抑制活性降低。当重组半胱氨酸蛋白酶抑制剂溶液在70℃保持5min时，抑制活性迅速降低。而当重组半胱氨酸蛋白酶抑制剂加热至90℃ 5min时，未检测到其抑制活性。重组中华鲟半胱氨酸蛋白酶抑制剂的抑制活性高于玉米种子中的半胱氨酸蛋白酶抑制剂（CPI），约1μg纯化的CIP可抑制0.278μg木瓜蛋白酶的活性。但重组半胱氨酸蛋白酶抑制剂的热稳定性低于玉米CPI。毕赤酵母重组半胱氨酸蛋白酶抑制剂的表达水平和活性高于大肠杆菌来源的。此外，毕赤酵母来源的重组半胱氨酸蛋白酶抑制剂更易于分离纯化。本文报道了在基于甲基营养型酵母的高效表达系统中生产重组鱼类半胱氨酸蛋白酶抑制剂，后续工作将聚焦于重组中华鲟半胱氨酸蛋白酶抑制剂抵抗鱼类疾病的功能，并探索半胱氨酸蛋白酶抑制剂作为食品添加剂在鱼糜加工过程中抑制半胱氨酸蛋白酶的价值。
