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

通过突变人γδ T细胞受体的结构测定分析非肽抗原识别机制

基本信息

批准号：
16590402
负责人：
TANAKA Yoshimasa
金额：
$ 1.98万
依托单位：
Kyoto University
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
2004
资助国家：
日本
起止时间：
2004 至 2005
项目状态：
已结题

项目摘要

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

在本研究中，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，然后以2.2Å分辨率测定晶体结构。实际上，编码 TCR γ 链和 δ 链胞外域的 cDNA 分别克隆并整合到基于 T7 的表达载体中。由于转染原始cDNA/载体的大肠杆菌未能产生重组蛋白，因此根据分子动力学计算去除了预测的阶梯环结构。此外，一些密码子被大肠杆菌喜欢的密码子取代，大肠杆菌密码子指数提高到0.65。基因修饰后，重组蛋白可以在大肠杆菌中以100 mg/l培养物或更高的浓度产生。将包涵体溶解在胍溶液中，用 DTT 处理，并使用快速稀释方法在基于精氨酸的缓冲液中重新折叠。透析后，根据pI判断，将蛋白质样品调至pH 5.5，并通过阳离子交换柱层析纯化，随后进行凝胶过滤。在筛选结晶时，使用了商业和内部制造的试剂盒。 1周后，在含有PEG4000的几种条件下出现晶体。条件改进后，晶体通过内部 X 射线发生器进行分析，分辨率为 3.0Å。然后，用SPring 8对晶体进行进一步分析。数据在工作站上进行处理，得到以下统计值：空间群； 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、细化； 15-1.90。如上所述，高分辨率解析了人γδT细胞受体的晶体结构

项目成果

期刊论文数量（45）

专著数量（0）

科研奖励数量（0）

会议论文数量（0）

专利数量（0）

ヒトγδ型T細胞の役割

人类 γδ T 细胞的作用

DOI：
发表时间：
2004
期刊：
BIO Clinica 19巻・5号
影响因子：
0
作者：
Ying Li;et al.;田中義正
通讯作者：
田中義正

Recognition and function of human gd T cells : Application for tumor immunotherapy

人gd T细胞的识别和功能：在肿瘤免疫治疗中的应用

DOI：
发表时间：
2005
期刊：
Cur.Immunol.Rev. 1
影响因子：
0
作者：
Seiji Yamashita;Yoshimasa Tanaka;Shunichi Tsutsumi;Hiroyuki Aburatani;Nagahiro Minato;Sigeo Ihara;Kenji Fukada;Yoshimasa Tanaka
通讯作者：
Yoshimasa Tanaka

Enhanced expression of programmed death-1 (PD-1)/PD-L1 in salivary glands of patients with Sjögren's syndrome.

DOI：
发表时间：
2005-11
期刊：
The Journal of rheumatology
影响因子：
0
作者：
M. Kobayashi;S. Kawano;S. Hatachi;Chiyo Kurimoto;Taku Okazaki;Yoshiko Iwai;T. Honjo;Yoshimasa Tanaka;N. Minato;T. Komori;S. Maeda;S. Kumagai
通讯作者：
M. Kobayashi;S. Kawano;S. Hatachi;Chiyo Kurimoto;Taku Okazaki;Yoshiko Iwai;T. Honjo;Yoshimasa Tanaka;N. Minato;T. Komori;S. Maeda;S. Kumagai

ヒトγδ型T細胞と抗腫瘍免疫

人类γδT细胞和抗肿瘤免疫