Ishii Y, Nakano T, Honma N, Yuyama N, Yamada Y, Watarai H, Tomura T, Sato M, Tsumura H, Ozawa T
Division of Immunobiology, La Jolla Institute for Allergy and Immunology, CA 92037, USA.
J Immunol Methods. 1995 Oct 12;186(1):27-36. doi: 10.1016/0022-1759(95)00126-u.
We have isolated a full length T cell receptor alpha chain (TCR alpha) cDNA derived from a bee venom phospholipase A2-specific mouse suppressor T cell hybridoma. A bacterial fusion expression system was constructed using rat calmodulin as a fusion partner for production of soluble TCR alpha. In this system, calmodulin-TCR alpha fusion protein was expressed at a high level in the soluble fraction of bacterial cell lysate, and could be purified by binding of calmodulin portion of the protein to phenyl-Sepharose. Using this system, fusion proteins containing a TCR alpha peptide corresponding to the complete extracellular region, V alpha-J alpha region or C alpha extracellular region were isolated. TCR alpha peptides were then released from the fusion proteins by digestion with thrombin which recognizes a linker sequence between calmodulin portion and TCR alpha segment. Polyclonal antibodies against constant region of TCR alpha chain (C alpha) were obtained by immunization of rabbits with the recombinant C alpha peptide. ELISA for TCR protein was established by using the polyclonal antibodies and the monoclonal antibody specific for C alpha region.
