人热休克蛋白转录因子1 DNA结合结构域的表达、纯化和晶体生长  

Expression,purification and Crystallization of Human Heat Shock Transcription Factor 1 DNA-Binding Domain

作　　者：回佳菡[1,2] 唐咏[3] 张春玲[1] 许凌峰[1] 李雪梅[1] 

机构地区：[1]中国科学院生物物理研究所 [2]沈阳农业大学生物技术学院辽宁沈阳110160 [3]沈阳农业大学生物技术学院

出　　处：《生物技术通讯》2006年第6期875-877,共3页Letters in Biotechnology

摘　　要：目的:获得大量热休克转录因子1(HSF1)DNA结合结构域(DBD)蛋白,用于晶体生长的三维结构解析。方法:将DBD基因片段克隆至原核表达载体pGEX-6P-1中并获得高效表达,经过Glutathione SepharoseTM 4B亲和层析、ResourceQ纯化后,蛋白纯度达到95%以上。结果:圆二色谱仪分析蛋白质的二级结构结果显示α螺旋占33%,β折叠占15%;采用悬滴气相扩散法得到了针状DBD晶体。结论:纯化的蛋白质与同源性达68%的Kluyveromyceslactis的DBD有相似的空间构象。获得的蛋白质晶体为进一步的三维结构解析奠定了基础。Objective： To obtain heat shock transcription factor I（HSF1） DNA-binding domain（DBD） and be used for X- ray diffraction experiments. Methods： The gene DBD was cloned into expression vector pGEX-6P-1 and got overexpression in the E.coli BL21 （DE3）. DBD protein was purified in a two-step procedure of Glutathione SepharoseTM 4B affinity and ion-exchange chromatography. Results： The secondary structure of protein was analyzed by circular dichroism spectroscopy, DBD contain 33% α-helix, 15% β-sheet. Single crystals of DBD were obtained by using hanging-drop vapourdiffusion method. Conclusion： The conformation of purified protein was similar with the DBD of Kluyveromyces lactis with 68% homologous secondary structure. Crystals were used for X-ray diffraction experiments.

关 键 词：人热休克蛋白转录因子1 DNA结合结构域 表达 蛋白质纯化 晶体生长 

分 类 号：Q786[生物学—分子生物学]