Engineering of Fc(1) and Fc(3) from human immunoglobulin G to analyse subclass specificity for staphylococcal protein A.
A system for production of recombinant Fc fragments of human IgG in Escherichia coli has been developed to allow for structural and functional studies of human Fc. The genes for the Fc fragments of human IgG subclasses 1 and 3, designated Fc(1) and Fc(3), were cloned from a human spleen cDNA library. The interactions to Staphylococcal protein A (SpA), a bacterial Fc receptor, that interacts with human IgG-Fc(1), but not with human IgG-Fc(3), were analyzed. To corroborate the involvement of amino acid residues in Fc, responsible for these differences in binding, 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 Fc proteins, covalently linked as dimers, were routinely obtained. SpA-binding analyses of all four Fc variants using biosensor technology, showed that Fc(1) and Fc(3(1)) interact 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 Fc expression system efficiently can be used in Fc engineering.
Jendeberg
L
L
Department of Biochemistry and Biotechnology, Royal Institute of Technology, Stockholm, Sweden.
Nilsson
P
P
Larsson
A
A
Denker
P
P
Uhlén
M
M
Nilsson
B
B
Nygren
P A
PA
D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000378:metabolism; D001483:Base Sequence; D015374:Biosensing Techniques; D006801:Humans; D007141:Immunoglobulin Fc Fragments / Q000737:chemistry / Q000378:metabolism*; D007074:Immunoglobulin G / Q000737:chemistry*; D008958:Models, Molecular; D008969:Molecular Sequence Data; D011485:Protein Binding; D011994:Recombinant Proteins; D013205:Staphylococcal Protein A / Q000378:metabolism*; D013329:Structure-Activity Relationship
D001426:Bacterial Proteins; C034284:IgG Fc-binding protein, Streptococcus; D007141:Immunoglobulin Fc Fragments; D007074:Immunoglobulin G; D011994:Recombinant Proteins; D013205:Staphylococcal Protein A