Preparation of Fcγ for addition to sulfhydryl-expressing ligands with minimal disturbance of the hinge

Abstract

We described previously a scheme for linking functionally intact human normal Fcγ1, via a thioether linkage emerging from its hinge, to any molecule expressing a free sulfhydryl group (SH). The scheme entails reducing the Fc to release four SH from the two inter-γ disulfide bonds (SS) in the hinge, blocking one SH by a stochastic alkylation, restoring by SS-interchange the inter-γ SS whose two SH are still available, and attaching a bismaleimide linker to the one remaining SH. One thereby obtains Fc with a single maleimide group (Fc-maleimide) for attachment to the SH-displaying partner. Restoration of the inter-γ SS is necessary if the final chimeric construct is to be able to activate the classical complement pathway. However, during this preparation of Fc-maleimide, there is apparently some SS-formation between non-homologous SH, so that not all hinges emerge with a reconstituted inter-γ SS. To reduce this error we have modified the preparative procedure after investigating an initial partial reduction of the hinge, and reviewing the conditions for stochastic alkylation. During partial reduction by dithiothreitol, the two hinge SS were cleaved apparently randomly: there was no evidence for one bond being more susceptible to reduction than the other, and little indication that the reduction of one bond enhanced the susceptibility of the other. By limiting reduction to an average of one SS per molecule, and alkylation to 0.8 SH per molecule, a final Fc-maleimide product is obtained in which most of the molecules have passed through the entire preparation with one of their hinge SS, and by inference much of the hinge conformation, remaining intact.