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

In a systematic study of the murine T-cell receptor UZ3-4, expressed and re folded from inclusion bodies in Escherichia coli, it was found that functio nal molecules can be obtained only under a very narrow set of conditions. T he refolded T-cell receptor UZ3-4 specifically recognizes its cognate pepti de (from mycobacterial Hsp60) in the context of H-2D(b), but not another pe ptide bound to H-2D(b), and the dissociation constant was determined by BIA core as 10(-4) M. Using T-cell receptor constructs comprising all extracell ular domains (V alpha C alpha and V beta C beta), found to be necessary for stability of the final product, significant amounts of native molecules we re obtained only if the intermolecular C alpha-C beta disulfide bridge bond was deleted, even though the interaction between the complete alpha and be ta-chain was determined to be very weak and fully reversible (K-D approxima te to 10(-7) to 10(-6) M). Fusion of Jun and Fos to the constant domains al so decreased the folding yield, because of premature association of interme diates leading to aggregation. Furthermore, only in a very narrow set of co ncentrations of oxidized and reduced glutathione, native disulfide bonds do minated. This shows that T-cell receptor domains are very prone to aggregat ion and misassociation during folding, compounded by incorrect disulfide bo nd formation. Once folded, however, the heterodimeric molecule is very stab le and could be concentrated to millimolar concentration. (C) 1999 Academic Press.