P450cam突变体蛋白表达、纯化与结晶的改进及其四突变体(F87L/Y96F/L244A/V247A)晶体结构

The Improved Procedure for The Expression, Purification and Crystallization of Mutants of Cytochrome P450cam and The Crystal Structure of The F87L/Y96F/L244A/V247A Mutant

在P4 5 0cam突变体蛋白纯化过程中使用 2 80nm/ 392nm双波长比值检测蛋白质纯度 ,采用 β 巯基乙醇对蛋白质进行还原性保护 ,有效地缩短了纯化进程 ,纯化效率相应提高 .以PEG 80 0 0为沉淀剂经悬滴汽相扩散法筛选得到适合衍射的P4 5 0cam四突变体 (F87L/Y96F/L2 4 4A/V2 4 7A)晶体 ,在Mar Research面探测器系统上收集了0 2 2nm分辨率的X射线衍射数据 .采用同晶差值傅立叶法解析结构 ,最后的晶体学R因子和Rfree分别为 0 197和 0 2 4 7,键长偏差为 0 0 0 177nm ,键角偏差为 1 96°.结构测定显示P4 5 0cam四突变体 (F87L/Y96F/L2 4 4A/V2 4 7A)和P4 5 0cam野生型的整体构象无重大变化 ,突变后活性口袋变大而疏水性增加 ,这与突变设计预期目标一致 .

The UV absorbance ratio (A(392)/A(280)) was developed to survey the purity of the P450cam mutant protein during the purification of the mutants protein. Thus the course of the purification became compact and the efficiency of the purification had been improved. beta-Mereaptoethenol had been developed to maintain the deoxidization of the protein during the purification. Crystals of the F87UY96F/1244A/V247A mutant were grown by vapor diffusion method. X-ray diffraction data were collected to 0.22 nm resolution on a Mar Research area detector in house. The structure was determined by Difference Fourier Method. The final crystallographic R factor and R-free factor are 0.197 and 0.247 respectively. The RMS deviations of bond length and angle of the mutants are 0.001 77 nm and 1.96degrees respectively. Structure comparison indicates that there is no obvious conformaional change between P450cam and the F87UY96F/L244A/V247A mutant. After mutation, the structure of the active pocket became larger but its hydrophobility increased, which are consistent with the aim of mutantion.