A soluble, single-chain T-cell receptor fragment endowed with antigen-combining properties.
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.
Novotny
J
J
Department of Macromolecular Modeling, Bristol-Myers Squibb, Princeton, NJ 08543-4000.
Ganju
R K
RK
Smiley
S T
ST
Hussey
R E
RE
Luther
M A
MA
Recny
M A
MA
Siliciano
R F
RF
Reinherz
E L
EL
D000595:Amino Acid Sequence; D000818:Animals; D000941:Antigens / Q000378:metabolism; D001665:Binding Sites; D003001:Cloning, Molecular; D003196:Computer Graphics; D004252:DNA Mutational Analysis; D019793:Fluorescein; D005452:Fluoresceins; D006801:Humans; D051379:Mice; D008958:Models, Molecular; D008969:Molecular Sequence Data; D011487:Protein Conformation; D011948:Receptors, Antigen, T-Cell / Q000737:chemistry* / Q000276:immunology; D011994:Recombinant Proteins; D016415:Sequence Alignment; D012995:Solubility; D013329:Structure-Activity Relationship
D000941:Antigens; D005452:Fluoresceins; D011948:Receptors, Antigen, T-Cell; D011994:Recombinant Proteins; D019793:Fluorescein