文章摘要
沙雷氏蛋白酶MP及其抑制剂LupI的复合物纯化及结晶
Purification and crystallization of complex of Serralysin-like MP and its inhibitor LupI
投稿时间:2019-04-30  修订日期:2019-05-24
DOI:
中文关键词: 沙雷氏蛋白酶  沙雷氏蛋白酶抑制剂  复合物  纯化  晶体
英文关键词: Serralysin-like  Serralysin-like inhibitor  Complex  Purification  Crystal
基金项目:国家实验室-鳌山科技计划(编号:2016ASKJ14)
作者单位E-mail
张琳桓 农业部极地渔业开发重点实验室中国水产科学研究院黄海水产研究所 上海海洋大学食品学院 962409424@qq.com 
王伟 农业部极地渔业开发重点实验室海洋药物与生物制品实验室  
孙晶晶 农业部极地渔业开发重点实验室海洋药物与生物制品实验室  
刘均忠 农业部极地渔业开发重点实验室海洋药物与生物制品实验室  
李尚勇 青岛大学医学院  
张洁 中国科学院天津工业生物研究所  
郝建华 农业部极地渔业开发重点实验室海洋药物与生物制品实验室江苏省海洋生物资源开发利用协同创新中心 haojh@ysfri.ac.cn 
摘要点击次数: 33
全文下载次数: 0
中文摘要:
      摘要:沙雷氏蛋白酶属于锌金属蛋白酶M10B亚家族,是一些细菌性疾病的关键致病因子。专一性沙雷氏蛋白酶抑制剂在体外可靶向抑制沙雷氏蛋白酶。通过沙雷氏蛋白酶抑制剂抑制沙雷氏蛋白酶,从而减弱产沙雷氏蛋白酶的细菌病原体的活性,成为疾病治疗的新思路。沙雷氏蛋白酶MP和其抑制剂LupI均来源于海洋微生物Flavobacterium sp. YS-80-122,本文分别纯化了蛋白酶MP、抑制剂LupI及MP-LupI的复合物,对MP-LupI复合物进行结晶条件筛选,成功在两种条件下获得MP-LupI复合物晶体,并经X-射线衍射收集到分辨率达2 ?的高质量衍射数据,其晶胞空间为P1 21 1,晶胞参数为a=51.66 ?、b=51.85 ?、c=102.14 ?、α=γ=90°、β=97.68°。
英文摘要:
      Abstract: Protease is a class of enzymes that hydrolyzes proteins by cutting protein peptide bonds in vivo, controlling protein size, composition, spatial conformation, and its final degradation. The physiological activities and diseases in the organism are closely related to proteases, such as digestion and absorption of food, blood coagulation, hemolysis, anti-inflammatory, blood pressure regulation, cell differentiation, cancer metastasis, and activation of physiologically active peptides. Serralysin-like belongs to the subfamily of zinc metalloprotease M10B and secretes by a variety of pathogenic Gram-negative bacteria. It is a key virulence factor for some diseases. Serralysin-like inhibitor can the inhibit target serralysin-like in vitro. The inhibition of bacterial virulence factors would present an antimicrobial strategy that is non-destructive, attenuating virulence mechanisms without directly challenging bacterial cell viability. The serralysin-like MP and its inhibitor LupI studied in this paper are all secreted by the marine microbial Flavobacterium sp. YS-80-122. We purified the protease MP, inhibitor LupI and MP-LupI comlex, respectively. The protease MP was purified by affinity chromatography while the inhibitor LupI was purified by size exclusion chromatography and ion exchange chromatography. After tested and concentrated, the two were mixed according to the equimolar ratio to obtain the MP-LupI complexes. Finally, purified MP-LupI complexes by size exclusion chromatography to obtain an electrophoresis-purified, single-purification sample of about 95 μL of MP-LupI complexes with a protein content of 20 mg/mL. Repeat the preparation of samples for subsequent experimental requirements. The MP-LupI complexes were crystallized and successfully obtained under two conditions: 0.1 M DL-Malic acid, pH 7.0, 12% (w/v) PEG 3350 to obtain a rhombohedral crystal of MP-LupI complex and 0.2 M NaCl, 0.1 M MES, pH 6.5, 10% (w/v) PEG 4000 to obtain an prism crystal of the MP-LupI complex. The X-ray diffraction data was collected from the Shanghai source to obtain high-quality diffraction data with a resolution of 2 ?. The unit cell space was P1 21 1. The unit cell parameters are a = 51.66 ?, b = 51.85 ?, c = 102.14 ?, α=γ=90° and β=97.68°.
相关附件:   图片  万方20190430(8.29%)  图片  审稿意见回复  万方20190515_审稿(8.00%)  附件1  附件2  万方20190523_审稿(7.89%)  附件1  万方20190524_审稿(7.90%)
View Fulltext   查看/发表评论  下载PDF阅读器
关闭