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J. Biol. Chem., Vol. 278, Issue 33, 30961-30970, August 15, 2003

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Production, Characterization, and Immunogenicity of a Soluble Rat Single Chain T Cell Receptor Specific for an Encephalitogenic Peptide^*

Rachel H. McMahan  , Lisa Watson ¶, Roberto Meza-Romero ¶, Gregory G. Burrows ¶ ||, Dennis N. Bourdette  ¶ and Abigail C. Buenafe  ¶ **

From the Department of ^¶Neurology and ^||Biochemistry and Molecular Biology, Oregon Health and Science University, Portland, Oregon 97201 and Neuroimmunology Research, Veterans Affairs Medical Center, Portland, Oregon 97201

The encephalitogenic rat T cell clone C14 recognizes the myelin basic protein 69–89 peptide in the context of the RT1B major histocompatibility complex (MHC) class II molecule. Modeling of the C14 TCR molecule indicated that previously identified CDR3 motifs are likely to be central to interaction with MHC class II-presented peptide. Here we report the cloning and expression of C14-derived single chain TCR (scTCR) molecules in an Escherichia coli expression system. The recombinant molecule consists of the V2 domain connected to the V8.2 domain via a 15-residue linker. Soluble C14 scTCR was purified using conventional chromatography techniques and refolded by a rapid dilution procedure. C14 scTCR was able to bind soluble rat MHC class II molecules bearing covalently coupled Gp-BP-(69–89) peptide, as analyzed using surface plasmon resonance. Immune recognition of the C14 scTCR protein as an antigen revealed that limited regions of the TCR may be more likely to induce responsiveness.

Received for publication, January 21, 2003 , and in revised form, May 20, 2003.

^* This work was supported by National Institutes of Health Grant NS35207 and NS041965, and National Multiple Sclerosis Society Grant RG3259A1. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Present address: Integrated Dept. of Immunology, National Jewish Medical and Research Center and the University of Colorado Health Sciences Center, 1400 Jackson St., Denver, CO 80206.

^** To whom correspondence should be addressed: Tykeson MS Research Laboratory UHS-46, Oregon Health and Science University, 3181 S.W. Sam Jackson Park Rd., Portland, OR 97201. Tel.: 503-494-4424; Fax: 503-494-9537; E-mail: buenafea{at}ohsu.edu.

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