PREPARATION OF SOLUBLE RECOMBINANT T-CELL RECEPTOR-ALPHA CHAIN BY USING A CALMODULIN FUSION EXPRESSION SYSTEM

We have isolated a full length T cell receptor alpha chain (TCR alpha) cDNA derived from a bee venom phospholipase A(2)-specific mouse suppr essor T cell hybridoma. A bacterial fusion expression system was const ructed using rat calmodulin as a fusion partner for production of solu ble TCR alpha. In this system, calmodulin-TCR alpha fusion protein was expressed at a high level in the soluble fraction of bacterial cell l ysate, and could be purified by binding of calmodulin portion of the p rotein to phenyl-Sepharose, Using this system, fusion proteins contain ing a TCR alpha peptide corresponding to the complete extracellular re gion, V alpha-J alpha region or C alpha extracellular region were isol ated. TCR alpha peptides were then released from the fusion proteins b y digestion with thrombin which recognizes a linker sequence between c almodulin portion and TCR alpha segment. Polyclonal antibodies against constant region of TCR alpha chain (C alpha) were obtained by immuniz ation of rabbits with the recombinant C alpha peptide. ELISA for TCR p rotein was established by using the polyclonal antibodies and the mono clonal antibody specific for C alpha region.