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.
出处
《中国生物工程杂志》
CAS
CSCD
北大核心
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
