运用基因克隆技术，以分离鉴定获得的蛋白质水解酶高活性类芽孢杆菌（ Paenibacillus lautus） CHN26菌株基因组DNA为模板，克隆鉴定了该菌株一种新型ATP⁃依赖型ClpP家族蛋白质水解酶PlclpP基因（585 bp），编码194个氨基酸，蛋白质分子量约为2．1×104。采用大肠杆菌（Eschericia coli） pET表达系统，构建了PlclpP基因表达质粒pET⁃28⁃PlclpP，并在大肠杆菌BL21中实现了重组PlClpP蛋白质的表达。利用组氨酸标签（ His⁃tag）亲和纯化法，获得了PlClpP纯化蛋白质，发现PlClpP可能与宿主菌未知伴侣分子形成蛋白质复合物。 PlClpP复合物具有ATP⁃依赖型酪蛋白水解酶活性，最适反应条件为40℃、pH7�0。表面活性剂强烈抑制PlClpP复合物的酶活，而常规丝氨酸蛋白酶抑制剂对其活性无抑制作用。%A novel ATP⁃dependent ClpP protease of Paenibacillus lautus CHN26 with high proteolytic activities which has recently been isolated and identified in our laboratory was cloned and expressed in this study. The clpP gene, designated as PlclpP, was 585 bp in size encoding 194 amino acids with a molecular weight of 2.1×104. The plasmid pET⁃28a⁃PlclpP was constructed, and the PlclpP gene was heterologously expressed in Escherichia coli BL21. The recombinant PlClpP protein was purified using Ni⁃NTA⁃His·Bind resin. PlClpP may form a complex with an unknown chaperone of E. coli BL21. The PlClpP complex showed a ATP⁃dependent proteolytic activity that reached the highest level at 40℃ with pH 7.0. In stead of conventional protease inhibitor, surfactant exhibited strong inhibition against PlclpP complex.
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