福氏痢疾杆菌组氨酸合成中双功能焦磷酸水解酶和磷酸核糖环化水解酶双功能蛋白的初步研究  

The Primary Study of the Bifunctional Phosphoribosyl-AMP cyclohydrolase/Phosphoribosyl-ATP Pyrophosphatase in Histidine Biosynthesis Pathway from Shigella flexneri 2α

作　　者：陆庆宇[1] 王其海[2] 张占钰[1] 袁道鹏[1] 高伟[1] 仓怀兴[2] 毕汝昌[2] 

机构地区：[1]北京林业大学理学院,北京100083 [2]中国科学院生物物理所,北京100101

出　　处：《中国生物工程杂志》2009年第4期56-60,共5页China Biotechnology

基　　金：国家林业局重点项目(2006-59);中科院创新工程资助项目

摘　　要：福志贺氏菌属是一类革兰氏阴性杆菌,是引起人类细菌性痢疾的主要致病源。以福志贺氏痢疾杆菌2a型301株全基因组为模板、以pET22b-(+)为载体、克隆了一个关键基因:组氨酸合成途径中双功能焦磷酸水解酶和磷酸核糖环化水解酶蛋白(简称:sf2088)。以BL21(DE3)为表达菌株,优化表达条件,获得了可溶表达的蛋白。经过亲和层析和分子筛层析获得目标蛋白。采用分析超速离心和动态光散射实验,对纯化后的蛋白进行稳定聚集状态的条件搜索,结果发现锌离子对稳定其聚合状态很重要。通过正交实验,获得蛋白保持稳定聚集态的条件。这对该酶的进一步研究奠定了基础。The metabolic pathway of histidine biosynthesis is significant for its various gene organizations and protein structures. HisI/E gene, that can express a bifunctional phosphoribosyl-AMP cyclohydrolase/ phosphoribosyl-ATP pyrophosphatase protein, which could catalyze the second and the third step in the histidine biosynthetic pathway, has been successfully cloned by using Shigella flexneri 2α （strain 301 ） genomic DNA as PCR temple. The protein was soluble expressed in pET22b vector, and purified by Ni-NTA cartridge and Superdex75 molecular filter. Then the soluble highly purified protein was characterized by preliminary crystal analysis, including dynamic light scattering, Analytical Uhracentrifugation and initially screened with Crystal Screens Ⅰ and Ⅱ and Index Screen （ Hampton Research）. The buffer of 50mmol/L Tris - HCl, pH 10.0, 300mmol/L NaCl, 0.05mmol/L ZnCl2 is the best solution for both the maintenance of oligomerization state of the protein and crystallization.

关 键 词：组氨酸合成途径 双功能蛋白 PR-ATP焦磷酸水解酶 PR-AMP解环酶 表达与纯化 

分 类 号：Q819[生物学—生物工程]