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.
Department of Molecular and Cell Biology, Institute of Medical Science, Foresterhill, Aberdeen, Scotland.
P E
PE
Molloy
W J
WJ
Harris
G
G
Strachan
C
C
Watts
C
C
Cunningham
D000595:Amino Acid Sequence; D000818:Animals; D015153:Blotting, Western; D004926:Escherichia coli / Q000235:genetics* / Q000276:immunology; D015964:Gene Expression Regulation, Bacterial; D006801:Humans; D051379:Mice; D008969:Molecular Sequence Data; D009154:Mutation; D010446:Peptide Fragments / Q000235:genetics* / Q000276:immunology; D016693:Receptors, Antigen, T-Cell, alpha-beta / Q000235:genetics* / Q000276:immunology; D013880:Thioredoxin-Disulfide Reductase / Q000235:genetics*
D010446:Peptide Fragments; D016693:Receptors, Antigen, T-Cell, alpha-beta; D013880:Thioredoxin-Disulfide Reductase