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. 1991 Oct 1;88(19):8646–8650. doi: 10.1073/pnas.88.19.8646

A soluble, single-chain T-cell receptor fragment endowed with antigen-combining properties.

J Novotny 1, R K Ganju 1, S T Smiley 1, R E Hussey 1, M A Luther 1, M A Recny 1, R F Siliciano 1, E L Reinherz 1
PMCID: PMC52566  PMID: 1924326

Abstract

A strategy for the production of small, soluble, single-chain T-cell receptor (scTCR) fragments that carry an intact TCR antigen-combining site is presented. The rationale is based on structural similarity between TCR and antibody molecules and use of computer modeling methods to derive a model structure of a human scTCR variable (V)-domain dimer. A gene encoding the RFL3.8 TCR protein, specific for the hapten fluorescein in the context of major histocompatibility complex class II and composed of one V alpha and one V beta domain joined via a flexible peptide linker, was assembled in an Escherichia coli plasmid. Subsequently, the protein was produced in a bacterial expression system, purified, refolded, and found to be poorly soluble at neutral pH in aqueous buffers. An inspection of the computer-generated V alpha-V beta domain model showed several surface exposed hydrophobic residues. When these were replaced by water-soluble side chains via site-directed mutagenesis of the corresponding gene, a soluble protein resulted and was shown to have antigen-binding properties equivalent to those of the intact TCR of the RFL3.8 T-cell clone. These results demonstrate the feasibility of obtaining TCR fragments endowed with antigen-combining properties by protein engineering in E. coli.

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