Prokaryotic production of the human T cell receptor Vbeta2 chain and binding to toxic-shock syndrome toxin-1.
The human T-cell receptor Vbeta2-, D- and J-encoding domains were PCR-amplified from MOLT-4 total cDNA and subcloned in Escherichia coli. The V/D/J fragment was subsequently transferred to a prokaryotic expression vector in frame with a polyhistidine-encoding prosequence which enabled us to affinity-purify the fusion protein with IMAC (immobilized metal-ion affinity chromatography [correction of chromatorgraphy]). Since the recombinant (re-) human T-cell receptor Vbeta2 fusion protein (Vbeta2 sol) produced in E.coli was found to be insoluble, purification was carried out under denaturating conditions. The purified and renatured re-protein, Vbeta2 sol, was immunoreactive with an anti-Vbeta2 monoclonal antibody in an ELISA assay. The specificity of Vbeta2 sol was shown by its binding in vitro to the staphylococcal superantigen TSST-1, but not to the Staphylococcus aureus exotoxin-1 (SEA).
Ericson
M L
ML
Laboratoire d'Immunogénétique Humaine, INSERM U396, Institut des Cordeliers, Paris, France.
Mooney
N A
NA
Charron
D J
DJ
D001427:Bacterial Toxins; D001483:Base Sequence; D002460:Cell Line; D003001:Cloning, Molecular / Q000379:methods; D017931:DNA Primers; D018076:DNA, Complementary; D004768:Enterotoxins / Q000378:metabolism*; D004926:Escherichia coli; D006801:Humans; D007700:Kinetics; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction; D011485:Protein Binding; D016693:Receptors, Antigen, T-Cell, alpha-beta / Q000096:biosynthesis* / Q000302:isolation & purification / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000302:isolation & purification; D011994:Recombinant Proteins / Q000096:biosynthesis* / Q000302:isolation & purification / Q000378:metabolism; D013211:Staphylococcus aureus; D018089:Superantigens / Q000378:metabolism
D001427:Bacterial Toxins; D017931:DNA Primers; D018076:DNA, Complementary; D004768:Enterotoxins; D016693:Receptors, Antigen, T-Cell, alpha-beta; D011993:Recombinant Fusion Proteins; D011994:Recombinant Proteins; D018089:Superantigens; C030107:enterotoxin F, Staphylococcal