Production of soluble single-chain T-cell receptor fragments in Escherichia coli trxB mutants.

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.

Expression and purification strategies for the production of single-chain antibody and T-cell receptor fragments in E. coli.

This work describes protocols for the production of single-chain antibody and T-cell receptor fragments in E. coli. A choice of methods is given for the purification of the recombinant fragments that rely on the use of either immunoaffinity or metal chelate affinity chromatography. The TCR fragments may have to be denatured and refolded before the fragments attain their proper conformation.

Spontaneous assembly of bivalent single chain antibody fragments in Escherichia coli.

The ability of immunoglobulin Fab and single chain (ScFv) fragments to penetrate effectively into tissue from the vascular system has made these molecules excellent candidates as drug delivery systems and imaging tools. This study investigates the use of single chain antibody fragment bacterial expression vectors as a possible strategy for the production of these molecules. We have modified the pSW1-VHD1.3-VKD1.3-TAG1 vector [Ward et al. (1989) Nature 341, 544-546] which originally, when expressed in E. coli, produced an Fab fragment. In an effort to improve the affinity of the parent vector product a novel single chain antibody construct which encodes a protein with anti-P. aeruginosa activity was generated using a 14 amino acid linker [Chaudhary et al. (1990) Proc. natn. Acad. Sci. U.S.A. 87, 1066-1070]. In addition to the heavy and light chain variable domain genes, our construct also contained the light chain kappa constant domain gene to aid purification of the fragments. To underline this difference from the conventional ScFv fragment we have described this protein as a ScAb. The ScAb generated had an antigen binding capacity similar to the parent anti-P. aeruginosa antibody but was superior to the recombinant anti-P. aeruginosa Fab fragment. On HPLC and non-denaturing gel electrophoresis analysis, the ScAb was found to exist in multimeric forms while the Fab fragment existed only as a single unit. Dimeric ScAb had a similar antigen binding profile to the parent antibody.