A bacterially expressed single-chain Fv construct from the 2B4 T-cell receptor.
A single-chain Fv construct of the 2B4 T-cell receptor has been made and expressed in Escherichia coli as bacterial inclusion bodies. After solubilization in 6 M guanidine hydrochloride and formation of mixed disulfides with glutathione, the protein was refolded by diluting out the denaturant and allowing intramolecular disulfide bridges to form by disulfide exchange. Approximately 65-100 mg of refolded protein was obtained from 1 liter of bacterial culture, an appreciable fraction of which was monomeric in nondenaturing solvents. This protein bound to three monoclonal antibodies specific for allotypic or idiotypic determinants on the native 2B4 variable region but did not bind several other anti-T-cell-receptor monoclonal antibodies that lacked such specificity. These experiments show that T-cell-receptor variable regions, like the V regions of antibodies, can form a well-behaved single-chain Fv molecule and provide large amounts of recombinant single-chain Fv T-cell receptor that can be used to study T-cell function.
Experimental Immunology Branch, National Cancer Institute, National Institutes of Health, Bethesda, MD 20892.
I
I
Kurucz
C R
CR
Jost
A J
AJ
George
S M
SM
Andrew
D M
DM
Segal
D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal; D000918:Antibody Specificity; D001483:Base Sequence; D002460:Cell Line; D003001:Cloning, Molecular; D010856:Columbidae; D003574:Cytochrome c Group / Q000235:genetics; D004797:Enzyme-Linked Immunosorbent Assay; D004926:Escherichia coli / Q000235:genetics; D006801:Humans; D006825:Hybridomas; D007700:Kinetics; D046911:Macromolecular Substances; D008969:Molecular Sequence Data; D009838:Oligodeoxyribonucleotides; D010957:Plasmids; D016133:Polymerase Chain Reaction / Q000379:methods; D011489:Protein Denaturation; D017510:Protein Folding; D011948:Receptors, Antigen, T-Cell / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D013601:T-Lymphocytes
D000911:Antibodies, Monoclonal; D003574:Cytochrome c Group; D046911:Macromolecular Substances; D009838:Oligodeoxyribonucleotides; D011948:Receptors, Antigen, T-Cell; D011994:Recombinant Proteins