PRODUCTION OF SOLUBLE SINGLE-CHAIN T-CELL RECEPTOR FRAGMENTS IN ESCHERICHIA-COLI TRXB MUTANTS

Antibodies and T cell receptors (TCR) both belong to the immunoglobuli n superfamily whose members are characterised by the possession of one or more immunoglobulin domains. The production of soluble single chai n 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 ch ain TCR produced in E. coil BL21 (DE3) and directed to the periplasm w as also insoluble and that this was in part due to the failure of the cell protein processing machinery to cleave the pe1B leader sequence. This problem was overcome by expressing the single chain TCR in the cy toplasm of E. coli which carry an inactive thioredoxin reductase gene. This strain allows the formation of disulphide bonds in the cell cyto plasm which we believe encourages the correct folding of the recombina nt protein. We have constructed both a human and mouse single chain TC R in these bacteria and demonstrated using BIAcore technology that the se molecules have folded in a conformation which allows their recognit ion 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. (C) 1998 Elsevier Science Ltd. All rights reserved.