鸡β2-微球蛋白及其硒代衍生物的制备和晶体学研究

Preparation of Selenomethionine-labelled Chicken β_2m and Its X-ray Crystallographic Studies

禽流感的暴发使得禽类免疫系统的研究显得愈加迫切,而以其他种属的同源β2-微球蛋白作为模板进行结构解析,不能解析鸡β2-微球蛋白(Chβ2m)的晶体结构。为了利用硒原子的反常散射获取Chβ2m晶体X射线衍射的相位信息,本研究以pET21a为表达载体,E.coli BL21(DE3)为宿主菌,在含有硒代甲硫氨酸的M9培养基中,用IPTG诱导表达硒代甲硫氨酸Chβ2m。包涵体经提取和稀释复性法复性之后,通过分子筛层析和Resource Q阴离子交换层析进行纯化,产物经SDS-PAGE检验纯度达98%以上。利用悬滴气相扩散法采用与Chβ2m蛋白晶体生长相同的条件,获得了衍生物可供衍射的晶体。通过北京同步辐射装置收集数据,最高分辨率至1.9。

It is becoming more and more important to study the avian immune system since the outbreak of avian influenza. In order to obtain the phase information of chicken β2-microglobulin （Chβ2m） crystal, the Chβ2m gene was inserted into pET21a vector and transformed to competent cell E. coli BL21 （DE3）. Selenomethionine-substituted protein was expressed as inclusion bodies in the selenomethionine culture medium. The inclusion bodies were refolded by diluted refolding method and purified with Superdex 75 and Resource Q. The protein was then crystallized using the hanging-drop vapour-diffusion method. Chβ2m crystal was obtained and used in data acquisition. Phase information was calculated using the muhiwavelength anomalous dispersion data obtained with a synchrotron-radiation source of Beijing Synchrotron Radiation Facility. The crystal belongs to P212121 space group with resolution of 1.9 A.