Hybrid immunoglobulin isotypes of identical specificity produced by genetic recombination in Escherichia coli and expression in lymphoid cells.
We have produced a series of hybrid IgG1.IgG2a mouse immunoglobulins with identical light chains (L) and variable regions to facilitate the identification of structural features associated with functional differences between immunoglobulin isotypes. Hybrid heavy chain (H) constant region gene segments were generated by genetic recombination in Escherichia coli between plasmids carrying mouse gamma 1 and gamma 2a gene segments. Crossovers occurred throughout these segments although the frequency was highest in regions of high nucleotide sequence homology. Eleven variant immunoglobulins produced by transfected hybridoma cell lines are assembled into H2L2 tetramers and properly glycosylated. In addition, all 11 immunoglobulins have identical antigen combining sites specific for the fluorescent hapten epsilon-dansyl-L-lysine. Protein A binding was used as a probe of the structural integrity of the Fc portion of these variant antibodies. Differences in protein A binding between IgG1 and IgG2a appear to be due to amino acid differences at positions 252 (Thr----Met) and 254 (Thr----Ser) of the heavy chain (EU numbering).
Schneider
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Department of Biological Sciences, Stanford University, California 94305.
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Yanofsky
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D000595:Amino Acid Sequence; D000818:Animals; D000918:Antibody Specificity; D001666:Binding Sites, Antibody; D003001:Cloning, Molecular; D004220:Disulfides; D004926:Escherichia coli / Q000235:genetics; D007132:Immunoglobulin Isotypes / Q000096:biosynthesis / Q000235:genetics*; D051379:Mice; D008969:Molecular Sequence Data; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics; D013205:Staphylococcal Protein A / Q000276:immunology; D013329:Structure-Activity Relationship
D004220:Disulfides; D007132:Immunoglobulin Isotypes; D011994:Recombinant Proteins; D013205:Staphylococcal Protein A