新型核苷碱基卤化酶AcmX的原核表达和结晶实验

Overexpression and crystallization of a novel nucleoside base halogenase AcmX

摘要:

在链霉菌中发现了一类能对核苷碱基进行特异位点卤化修饰的新型卤化酶，可用于核苷类药物的卤代优化。对新型核苷碱基卤化酶晶体结构的解析，是阐明其生物催化的机理，建立生物催化卤化制备核苷类药物的最直接手段。为了获得一个卤化酶AcmX的晶体结构，首先通过大肠杆菌原核表达制备AcmX蛋白样品，但只能得到没有活性的AcmX的蛋白包涵体。通过构建AcmX、分子伴侣蛋白共表达系统，注意控制合适的分子伴侣蛋白表达水平，并通过高分辨率的分子筛凝胶层析步骤，去除混入AcmX样品的分子伴侣蛋白GroEL，摸索出表达和纯化高质量AcmX蛋白样品的方法，并将蛋白样品成功用于结晶实验。这不仅为解析卤化酶AcmX的晶体结构打下坚实的基础，还可将本研究的方法用于其他表达困难的蛋白的制备。

Abstract:

Most recently a new group of regioselective adenine halogenases AcmX and AcmY have been found in Streptomyces, which can be employed to develop the halide substitution of nucleoside-like drugs. The crystal structures of this new family of halogenase are the basis to elucidate the catalytic mechanism and biosynthesize nucleoside-like drugs with halogen modifications. In order to solve the structure of AcmX of this new halogenase family, AcmX was overexpressed in Escherichia coli but was initially as the inactive inclusion body. Thus we constructed a dual expression system for AcmX and the chaperone plasmid pGro7, which promotes AcmX to fold properly by controlling the expression level of the chaperone plasmid. The chaperone protein is inclined to co-purify with the target protein but can be removed by a step of gel filtration chromatography, so that the protocol can be established to obtain AcmX in high quality, leading to the successful crystallization. This study will facilitate X-ray diffraction data collection and structure determination for AcmX and provide a useful protocol for the other difficult cases in expression and purification.