Analysis of mechanism for nonpeptide antigen recognition by structural determination of mutated human γδ T cell receptor

本研究将人γδ T细胞受体的胞外结构域在大肠杆菌中表达，重新折叠在一起并结晶，然后在2.2Å分辨率下测定晶体结构。在实践中，编码TCR γ链和δ链胞外结构域的cdna分别被克隆并整合到基于t7的表达载体中。由于原cDNA/载体转染大肠杆菌后无法产生重组蛋白，因此根据分子动力学计算，将预测的阶跃环结构去除。此外，一些密码子被大肠杆菌偏爱的密码子取代，大肠杆菌密码子指数提高到0.65。基因修饰后的重组蛋白在大肠杆菌中培养量达到100mg /l或更高。将包涵体溶解在胍溶液中，用DTT处理，并使用快速稀释法在精氨酸基缓冲液中折叠。透析后的蛋白样品经pl判断pH为5.5，经阳离子交换柱层析纯化，凝胶过滤。在筛选结晶，商业和内部自制试剂盒使用。1周后，在几种含有PEG4000的条件下出现晶体。改善条件后，用内部x射线发生器对晶体进行分析，得到3.0Å分辨率。然后用spring8对晶体进行进一步分析。在工作站对数据进行处理，得到以下统计值：空间组；P21，单位；60.82, 77.35, 100.12, 90.00, 95.00，和90.00，反射；314、713，独立反思；65, 401，决议；1.9、完整性；91.4、冗余；4.8、精细化；15 - 1.90。如上所述，以高分辨率解析了人γδ T细胞受体的晶体结构

In the present study, the extracellular domains of a human γδ T cell receptor were expressed in E.coli, refolded together, and crystallized, then the crystal structure was determined at a 2.2Å resolution. In practice, cDNAs encoding the extracellular domains of TCR γ- and δ-chains were separately cloned and incorporated into a T7-based expression vector. Since E.coli transfected with the original cDNA/vector failed to produce the recombinant proteins, the predicted step loop structure was removed on the basis of molecular dynamics caliculation. In addition, several codons were replaced by E.coli-favored ones and the E.coli codon index was increased up to 0.65. After the gene modification, the recombinant proteins could be produced in E.coli at 100 mg/l culture or more. The inclusion bodies were dissolved in guanidine solution, treated with DTT and refolded in an arginine-based buffer using a rapid dilution method. After dialysis, the protein sample was brought to pH 5.5 judging from pl and purified on cation-exchange column chromatography, followed by gel filtration. In screening for crystallization, commercial and in-house made kits were used. After 1 week, crystals appeared in several conditions containing PEG4000. After improvement of the conditions, the crystals were analyzed by the in-house X-ray generator, which gave a 3.0Å resolution. Then, the crystals were further analyzed by SPring 8. The data were processed on workstation and the following statistical values were obtained : space group ; P21, unit ; 60.82, 77.35, 100.12, 90.00, 95.00, and 90.00, reflection ; 314, 713, independent reflection ; 65, 401, resolution ; 1.9, completeness ; 91.4, redundancy ; 4.8, and refinement ; 15-1.90. As described above, crystal structure of human γδ T cell receptor was solved at a high resolution