重组肺炎链球菌甘油脱氢酶活性及晶体培养的初步分析

甘油脱氢酶能够氧化胞内甘油为代谢活动提供能量，并参与调控与宿主粘附相关细菌蛋白质的表达，是潜在的抗菌药物靶点. 本文利用大肠杆菌BL21原核表达肺炎链球菌甘油脱氢酶，获得大量上清表达的目的蛋白. 再用Ni-NAT亲和层析、DEAE离子交换层析以及Superdex 200分子筛进行纯化. 经数据拟合，证实该酶在溶液中以同源二聚体形式存在. 光谱实验证实该重组表达甘油脱氢酶仍具有氧化甘油生成1, 3-二羟基丙酮的活性. 用Hampton试剂盒初筛晶体及棋盘法优化晶体，在0.1 mol/L Bicine PH 9.6，13% PEG MME 5000，0.2 mol/L KSCN，4% Dioxone条件下，得到了晶型好且有一定衍射能力的单晶，为解析肺炎链球菌甘油脱氢酶的三维结构及研究结构与酶活之间的关系奠定了基础.

A Preliminary Study of the Bioactivity and Crystallization of the Recombinant Glycerol Dehydrogenase from Streptococcus pneumoniae

Glycerol dehydrogenase (GldA) hydrolyzes glycerol to provide energy for some metabolic process. In addition, GldA regulates the expression of some pathogen proteins relating to the adhesion to host. Consequently, the pathogen GldA was considered as a potential target of novel antibacterial agent drugs. Here, the expression, purification, bioactivity determination and crystallization of the GldA of Streptococcus pneumoniae (S. pneumoniae) are reported. The recombinant protein of GldA was expressed by E. coli BL21 in supernatant and purified by Ni-NAT column, DEAE fast flow and gel-filtration Superdex 200. The recombinant enzyme exhibited as homodimer in solution and could hydrolyze glycerol to dihydroxyacetone. Initial crystallization screening was carried out using Hampton kits by the sitting-drop method. Single crystal was obtained by using the hanging drop vapour diffusion method in 0.1 mol/L Bicine pH9.6, 13% PEG MME 5000, 0.2 mol/L KSCN and 4% Dioxone. These results provide some key basements to study the relationship between the three-dimensional structure and bioactivity of GldA.