Molloy P E, Harris W J, Strachan G, Watts C, Cunningham C
Department of Molecular and Cell Biology, Institute of Medical Science, Foresterhill, Aberdeen, Scotland.
Mol Immunol. 1998 Feb;35(2):73-81. doi: 10.1016/s0161-5890(98)00019-4.
Antibodies and T cell receptors (TCR) both belong to the immunoglobulin superfamily whose members are characterised by the possession of one or more immunoglobulin domains. The production of soluble single chain antibody fragments in Escherichia coli has, in recent years, become a routine laboratory procedure. In contrast, the production of T cell receptors in bacteria has remained problematic as the majority of the recombinant protein is insoluble. In this paper we show that single chain TCR produced in E. coli BL21 (DE3) and directed to the periplasm was also insoluble and that this was in part due to the failure of the cell protein processing machinery to cleave the pelB leader sequence. This problem was overcome by expressing the single chain TCR in the cytoplasm of E. coli which carry an inactive thioredoxin reductase gene. This strain allows the formation of disulphide bonds in the cell cytoplasm which we believe encourages the correct folding of the recombinant protein. We have constructed both a human and mouse single chain TCR in these bacteria and demonstrated using BIAcore technology that these molecules have folded in a conformation which allows their recognition by conformational specific ligands. In addition, we have used one of our soluble single chain TCR preparations to isolate a TCR specific Fab molecule from a phage antibody library.
抗体和T细胞受体（TCR）都属于免疫球蛋白超家族，其成员的特征是拥有一个或多个免疫球蛋白结构域。近年来，在大肠杆菌中生产可溶性单链抗体片段已成为常规实验室操作。相比之下，在细菌中生产T细胞受体仍然存在问题，因为大多数重组蛋白是不溶性的。在本文中，我们表明在大肠杆菌BL21（DE3）中产生并导向周质的单链TCR也是不溶性的，部分原因是细胞蛋白质加工机制未能切割pelB前导序列。通过在携带无活性硫氧还蛋白还原酶基因的大肠杆菌细胞质中表达单链TCR，克服了这个问题。该菌株允许在细胞质中形成二硫键，我们认为这有助于重组蛋白的正确折叠。我们已经在这些细菌中构建了人源和鼠源单链TCR，并使用BIAcore技术证明这些分子已折叠成一种构象，使其能够被构象特异性配体识别。此外，我们已经使用我们的一种可溶性单链TCR制剂从噬菌体抗体库中分离出一种TCR特异性Fab分子。
