黑曲霉内切葡聚糖酶AnCel5A的表达纯化与晶体优化

Expression,Purification and Crystallization of a Glycoside Hydrolase Family 5 Endoglucanase(AnCel5A) from Aspergillus niger

来源于黑曲霉(Aspergillus niger)的β-1,4-内切葡聚糖酶(AnCel5A)是糖苷水解酶第5家族成员,水解纤维素分子无定形区的β-1,4-糖苷键,在纤维素的降解过程中发挥关键作用。本研究通过对AnCel5A的结晶条件初筛,获得并优化AnCel5A晶体以期解析其蛋白质晶体结构。将截去N端信号肽的AnCel5A蛋白在Pichia pastoris X33/pPICZαA中进行表达,发酵液上清透析除盐并进行Endo H去糖基化酶切,后续依次通过离子交换层析、疏水层析,获得高纯度蛋白。通过坐滴气相扩散法对AnCel5A的结晶条件进行初筛,在CryoI screen kit (A6)孔中长出了初始晶体,经优化获得了更好的晶体。蛋白晶体于台湾新竹同步辐射中心(NSRRC)BL15A1线站进行了X射线衍射数据收集,结果显示其分辨率达到1.8?。

The β-1,4-endoglucanase from Aspergillus niger(AnCel5 A) is a cellulase which is classified to glycoside hydrolases subfamily(GH) 5. It can hydrolyze the amorphous region of cellulose long chains by endo-action,which makes it one of the key enzymes in cellulose hydrolysis.This research aims at determination the crystal structure of AnCel5 A by screening and optimization its protein crystals. The signal peptide truncated AnCel5 A is successfully expressed in P. pastoris X33. Purified proteins are obtained by using ion-exchange and hydrophobic chromatography. The crystal screen of AnCel5 A is completed by using sitting-drop vapor diffusion method and screen kits bought from the Hampton Research company. Initial crystals are found in the reservoir solution of cryoI kit(A6). After further optimization,better crystals are obtained. The crystals diffracted to 1.8 ? and dataset is collected at beam line BL15A1 of the National Synchrotron Radiation Research Center(NSRRC,Hsinchu,Taiwan).