Development of methodologies for the provision of soluble T cell receptors
The aim of this work presented here was to develop a method which would allow the production of soluble T-cell receptor (TCR) fragments in E. coli. to this end we have isolated and sequenced the α and β chain variable region genes from a human cell line specific for tetanus toxin. Ligation-anchored polymerase chain reaction (PCR) and rapid amplification of cDNA ends (RACE) techniques were used, and the α and β variable chain genes were successfully isolated using a RACE method. Having successfully cloned and sequenced the α and β variable chain genes from the human tetanus-toxin-specific D3 TCR cell line these genes had to be 'rescued' and linked together to form a single chain TCR (scTCR) for periplasmic expression. Since attempts to direct expression of the D3 TCR to the periplasm of E. coli were unsuccessful, it was therefore decided that we should try and direct protein expression to the cytoplasm. A cytoplasmic expression vector was constructed from pD3-TCR by deletion of the leader sequence and was called pD4-TCR yielding D4 scTCR protein. We have successfully produced and purified soluble disulphide-bonded D4 scTCR and 114 scTCR from the cytoplasm of the trxB- E. coli strain AD494 with expression yields of ~200μg/L. Greater than 50% of the scTCRs produced in the E. coli AD494 was disulphide bonded. We have successfully produced anti-D4 scTCR Fabs by phage display, and attempted to show they could bind to native D3 T-cells. Due to non-specific binding of phage, we could not demonstrate that the phage specifically recognised D3 T-cells; however, soluble Fab PM1 derived from the third-round panning could be shown to bind to D4 scTCR using BIAcore analysis. The PM1 Fab recognised disulphide-bonded D4 scTCR better than non-disulphide-bonded D4 scTCR, perhaps indicating that it could be a conformational-specific Fab.