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

J. Novotny, R. K. Ganju, S. T. Smiley, R. E. Hussey, M. A. Luther, M. A. Recny, R. F. Siliciano, E. L. Reinherz

Research output: Contribution to journalArticlepeer-review


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(α) and one V(β) 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(α)-V(β) 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.

Original languageEnglish (US)
Pages (from-to)8646-8650
Number of pages5
JournalProceedings of the National Academy of Sciences of the United States of America
Issue number19
StatePublished - 1991


  • Escherichia coli expression
  • immunoglobulin domains
  • protein engineering
  • variable regions

ASJC Scopus subject areas

  • General


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