Schodin B A, Schlueter C J, Kranz D M
Department of Biochemistry, University of Illinois, Urbana 61801, USA.
Mol Immunol. 1996 Jun;33(9):819-29. doi: 10.1016/0161-5890(96)00038-7.
The diversity and domain structure of alpha beta T cell receptors (TCR) are similar to immunoglobulins based on sequence homologies, but the three-dimensional structure of the alpha beta-heterodimer has not been solved. To begin structure/function studies, we have compared the properties of a soluble single-chain V alpha V beta TCR (scTCR) expressed in three E. coli systems. The V alpha and V beta regions were expressed with pelB or ompA signal sequences or as a thioredoxin fusion protein. The scTCRs were detected only in the insoluble fraction of the cells and could be solubilized in guanidine and renatured to obtain properly folded scTCR from each system. Only a small fraction (1-5%) of the ompA and pelB scTCRs folded properly. In contrast, the thioredoxin fusion protein exhibited high total yields and a solubility that was ten times higher than the other scTCRs. The thioredoxin fusion protein also bound specifically to the peptide/MHC ligand with a KD of approximately 0.7 microM, as shown by a competitive inhibition assay with Fab fragments that recognize the MHC complex. Furthermore, estimates from saturation binding with antibodies that react with the native TCR indicated that up to 80% of the thioredoxin fusion protein was in the properly folded form. The improved yield, solubility, and binding activity of the thioredoxin-scTCR should make it useful for various structure/ function studies.
基于序列同源性，αβ T细胞受体（TCR）的多样性和结构域结构与免疫球蛋白相似，但αβ异二聚体的三维结构尚未解析。为了开展结构/功能研究，我们比较了在三种大肠杆菌系统中表达的可溶性单链VαVβ TCR（scTCR）的特性。Vα和Vβ区域与pelB或ompA信号序列一起表达，或作为硫氧还蛋白融合蛋白表达。scTCR仅在细胞的不溶性部分中检测到，并且可以在胍中溶解并复性，以从每个系统中获得正确折叠的scTCR。只有一小部分（1-5%）的ompA和pelB scTCR正确折叠。相比之下，硫氧还蛋白融合蛋白表现出高总产量，其溶解度比其他scTCR高十倍。如用识别MHC复合物的Fab片段进行的竞争性抑制试验所示，硫氧还蛋白融合蛋白还以约0.7μM的KD特异性结合肽/MHC配体。此外，用与天然TCR反应的抗体进行饱和结合估计表明，高达80%的硫氧还蛋白融合蛋白处于正确折叠的形式。硫氧还蛋白-scTCR产量、溶解度和结合活性的提高使其对各种结构/功能研究有用。
