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 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.
Ishii
Y
Y
Division of Immunobiology, La Jolla Institute for Allergy and Immunology, CA 92037, USA.
Nakano
T
T
Honma
N
N
Yuyama
N
N
Yamada
Y
Y
Watarai
H
H
Tomura
T
T
Sato
M
M
Tsumura
H
H
Ozawa
T
T
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002147:Calmodulin / Q000737:chemistry; D003001:Cloning, Molecular; D017931:DNA Primers / Q000737:chemistry; D018076:DNA, Complementary / Q000235:genetics; D006825:Hybridomas; D051379:Mice; D008969:Molecular Sequence Data; D010741:Phospholipases A / Q000276:immunology; D054467:Phospholipases A2; D011817:Rabbits; D051381:Rats; D016693:Receptors, Antigen, T-Cell, alpha-beta / Q000737:chemistry*; D011993:Recombinant Fusion Proteins / Q000737:chemistry; D012995:Solubility; D050378:T-Lymphocytes, Regulatory / Q000737:chemistry
D002147:Calmodulin; D017931:DNA Primers; D018076:DNA, Complementary; D016693:Receptors, Antigen, T-Cell, alpha-beta; D011993:Recombinant Fusion Proteins; D010741:Phospholipases A; D054467:Phospholipases A2