人口腔癌症缺失相关蛋白(DOC-1R)的表达、纯化和晶体生长  

EXPRESSION,PURIFICATION AND CRYSTALLIZATION OF HUMAN DOC-1R

作　　者：王杰[1] 程中军[1] 龚为民[1] 

机构地区：[1]中国科学技术大学生命科学学院

出　　处：《生物物理学报》2006年第3期212-216,共5页Acta Biophysica Sinica

基　　金：国家自然科学基金项目(10490193)~~

摘　　要：口腔癌缺失(DeletedinOralCancer-1,DOC-1)基因是近年来被证实的口腔癌中具有抑癌作用的基因。1999年,研究人员通过酵母双杂交实验又发现了与DOC-1相关的另一候选抑癌基因DOC-1R(DOC-1related)。以往的很多实验表明,这两个蛋白无论序列还是功能上都非常相似。然而,其三维结构以及与其他重要蛋白相互作用的机制一直还不清楚,PDB库中也未见其相关同源结构的报道。作者将人DOC-1R基因的cDNA片段克隆至原核表达载体pET-22b(+)中,通过IPTG诱导获得高效表达,再经过Ni-NTA亲和层析和Superdex75层析柱纯化,获得了纯度达到96%以上的蛋白。质谱分子量测定显示DOC-1R的分子量为14091.23Da,与理论分子量基本一致;动态光散射实验显示蛋白均一性高达99.0%,可用于晶体生长;采用悬滴气相扩散法筛选,在多个条件下得到了DOC-1R的微晶。为DOC-1R的三维结构解析奠定了坚实的基础。DOC-1（Deleted in Oral Cancer-1） is a growth suppressor gene identified first in oral cancer. DOC-1R（DOC-1 related） gene is another tumor suppressor gene which was cloned in 1999. Recent studies suggested that DOC-1 and DOC-1R share similarity in both of their sequences and functions. However, there is no homology structure found in PDB. Human doc-1r gene was cloned into expression vector pET-22b（＋） and over-expressed in E.coli strain BL21 （DE3）. Human DOC-1R protein was purified in a two-step procedure of Ni-NTA affinity followed by Superdex 75 chromatography. Over 98% of purified DOC-1R proteins was finally got for structural and functional research. Mass Spectrum identified that the exact molecular weight of DOC-1R is 14091.23 Da; Dynamic light scattering assay showed that its homogenesis was over 99.0%. The crystals of DOC-1R were obtained by using hanging-drop vapor-diffusion method.

关 键 词：口腔癌症缺失 DOC-1R 表达 纯化 质谱 动态光散射 晶体生长 

分 类 号：Q73[生物学—分子生物学]