Research output: Contribution to Journal/Magazine › Journal article
A bacterially expressed single-chain Fv construct from the 2B4 T-cell receptor. / Kurucz, I.; Jost, C. R.; George, A. J. T. et al.
In: Proceedings of the National Academy of Sciences of the United States of America, Vol. 90, No. 9, 1993, p. 3830-3834.Research output: Contribution to Journal/Magazine › Journal article
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TY - JOUR
T1 - A bacterially expressed single-chain Fv construct from the 2B4 T-cell receptor.
AU - Kurucz, I.
AU - Jost, C. R.
AU - George, A. J. T.
AU - Andrew, S. M.
AU - Segal, D. M.
PY - 1993
Y1 - 1993
N2 - 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.
AB - 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.
M3 - Journal article
VL - 90
SP - 3830
EP - 3834
JO - Proceedings of the National Academy of Sciences of the United States of America
JF - Proceedings of the National Academy of Sciences of the United States of America
SN - 0027-8424
IS - 9
ER -