ENGINEERING OF FC(1) AND FC(3) FROM HUMAN-IMMUNOGLOBULIN-G TO ANALYZESUBCLASS SPECIFICITY FOR STAPHYLOCOCCAL PROTEIN-A

A system for production of recombinant Fc fragments of human IgG in Es cherichia coli has been developed to allow for structural and function al studies of human Fc. The genes for the Fc fragments of human IgG su bclasses 1 and 3, designated Fc(1) and Fc(3), were cloned from a human spleen cDNA library. The interactions to Staphylococcal protein A (Sp A), a bacterial Fc receptor, that interacts with human IgG-Fc(1), but not with human IgG-Fc(3), were analyzed. To corroborate the involvemen t of amino acid residues in Pc, responsible for these differences in b inding, two Fc variants were constructed; Fc(1(3)) and Fc(3(1)), each containing an isotypic dipeptide substitution. Production levels in E. coli of 1-10 mg/l of secreted and Pc Fc proteins, covalently linked a s dimers, were routinely obtained. SpA-binding analyses of all four Pc variants using biosensor technology, showed that Fc(1) and Fc(3(1)) i nteract with SpA, while Fc(3) and Fc(1(3)) lack detectable SpA binding . The rendered SpA binding of the Fc variant Fc(3(1)), is concluded to result from the introduced dipeptide substitution (R435H, F436Y). The results demonstrate that the Pc expression system efficiently can be used in Fe engineering.