HIGH-LEVEL PRODUCTION OF A SECRETED, HETERODIMERIC ALPHA-BETA MURINE T-CELL RECEPTOR IN ESCHERICHIA-COLI

For structural studies, high-level production of properly folded, disu lfide-linked, unglycosylated protein in E. coli is an attractive alter native to production in eukaryotic systems. We describe here the produ ction of heterodimeric, murine D10 T-cell receptor (sD10TCR) in E. col i as a secreted leucine zipper (LZ) fusion protein. Two genes, one (al pha-acid) encoding the alpha-chain variable and constant domains (V al pha and C alpha) of D10 TCR fused to an LZ 'acid' encoding sequence an d the other (beta-base) encoding the beta-chain variable and constant domains (V beta and C beta) fused to an LZ 'base' encoding sequence, w ere co-expressed from a bacteriophage T7 promoter as a dicistronic mes sage. Secreted alpha-acid and beta-base proteins formed proper inter- and intra-chain disulfide bonds in the periplasm, bypassing the need f or in vitro protein refolding. Complementary LZ sequences facilitated the formation of alpha beta heterodimers. sD10TCR-LZ was purified by a ffinity chromotography using a D10 TCR clonotype-specific monoclonal a ntibody (mAb 3D3). Typical yields of purified protein were 4-5 mg/l of culture. Purified sD10TCR-LZ was reactive with a panel of conformatio nally sensitive TCR-specific monoclonal antibodies, consistent with it s conformational integrity and appeared to be suitable for structural studies by X-ray crystallography or NMR spectroscopy. (C) 1997 Elsevie r Science B.V.