Bacterially expressed human Fc gamma RIIb is soluble and functionally active after in vitro refolding

A recombinant soluble form of the human Fc gamma receptor was produced by e ngineering a cDNA construct containing the extracellular part of the mature protein. After expression in bacteria as inclusion body, the polypeptide w as highly purified and was refolded in vitro with a method that was develop ed for the renaturation of immunoglobulin fragments. With this method oxida tion of the disulfide bridges within the domains of the protein is done in the presence of an artificial 'chaperone' which protects the polypeptide mo lecules from unwanted protein-protein interactions thereby inhibiting the i ncorrect oxidation of the SH-groups, and misfolding of the protein. The ref olded recombinant soluble Fc gamma RIIb showed several characteristics of t he native receptor: (i) it was recognized by a series of monoclonal antibod ies specific for, and in most cases produced against the native cell-surfac e receptor; (ii) it is bound to its ligand (the Fc-region of different immu noglobulins) under very diverse conditions; and (iii) it is competed strong ly and specifically with the native cell surface receptor for both ligand a nd antibody binding in experiments with distinct read-outs; (iv) monoclonal antibodies produced against the recombinant protein specifically recognize d Fc gamma RIIb on different cells. From these data it was concluded that t he recombinant soluble Fc-receptor was in a native, functionally active for m, and its function was not affected by the lack of glycosylation. (C) 2000 Elsevier Science B.V. All rights reserved.