[1]龚丽,李云霞.一种新型ATP-依赖型ClpP家族蛋白质水解酶PlclpP基因的克隆、表达和酶学特性[J].江苏农业学报,2015,(06):1424-1429.[doi:doi:10.3969/j.issn.1000-4440.2015.06.036]
 GONG Li,LI Yun-xia.Cloning, expression and characterization of a novel PlclpP gene encoding ATP-dependent ClpP protease[J].,2015,(06):1424-1429.[doi:doi:10.3969/j.issn.1000-4440.2015.06.036]
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一种新型ATP-依赖型ClpP家族蛋白质水解酶PlclpP基因的克隆、表达和酶学特性()
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江苏农业学报[ISSN:1006-6977/CN:61-1281/TN]

卷:
期数:
2015年06期
页码:
1424-1429
栏目:
加工贮藏·质量安全
出版日期:
2015-12-31

文章信息/Info

Title:
Cloning, expression and characterization of a novel PlclpP gene encoding ATP-dependent ClpP protease
作者:
龚丽李云霞
(农业部水产品贮藏保鲜质量安全风险评估重点实验室<上海>,上海海洋大学食品科学与技术学院,上海201306)
Author(s):
GONG LiLI Yun-xia
(Laboratory of Quality and Safety Risk Assessment for Aquatic Products on Storage and Preservation , Ministry of Agriculture, College of Food Science and Technology, Shanghai Ocean University, Shanghai 201306, China)
关键词:
ClpP家族蛋白质水解酶芽孢杆菌基因克隆基因表达酶学特性
Keywords:
ClpP proteasePaenibacillus lautusgene cloninggene expressionenzymatic characteristic
分类号:
Q78
DOI:
doi:10.3969/j.issn.1000-4440.2015.06.036
文献标志码:
A
摘要:
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×104。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。
Abstract:
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×104. 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.

参考文献/References:

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备注/Memo

备注/Memo:
收稿日期:2015-05-08 基金项目:上海市科委项目(09320503600) 作者简介:龚丽(1989-),女,上海人,硕士研究生,主要从事食品质量与安全研究。(E-mail) emo_jun@163.com 通讯作者:李云霞,(E-mail)liyunxia925@qq.com
更新日期/Last Update: 2015-12-31