Folding, heterodimeric association and specific peptide recognition of a murine alphabeta T-cell receptor expressed in Escherichia coli

In a systematic study of the murine T-cell receptor UZ3-4, expressed and refolded from inclusion bodies in Escherichia coli, it was found that functional molecules can be obtained only under a very narrow set of conditions. The refolded T-cell receptor UZ3-4 specifically recognizes its cognate peptide (from mycobacterial Hsp60) in the context of H-2Db, but not another peptide bound to H-2Db, and the dissociation constant was determined by BIAcore as 10(-4) M. Using T-cell receptor constructs comprising all extracellular domains (ValphaCalpha and VbetaCbeta), found to be necessary for stability of the final product, significant amounts of native molecules were obtained only if the intermolecular Calpha-Cbeta disulfide bridge bond was deleted, even though the interaction between the complete alpha and beta-chain was determined to be very weak and fully reversible (KD approximately 10(-7) to 10(-6) M). Fusion of Jun and Fos to the constant domains also decreased the folding yield, because of premature association of intermediates leading to aggregation. Furthermore, only in a very narrow set of concentrations of oxidized and reduced glutathione, native disulfide bonds dominated. This shows that T-cell receptor domains are very prone to aggregation and misassociation during folding, compounded by incorrect disulfide bond formation. Once folded, however, the heterodimeric molecule is very stable and could be concentrated to millimolar concentration.