Bifunctional hybrids between the variable domains of an immunoglobulin and the maltose-binding protein of Escherichia coli: production, purification and antigen binding.
Hybrids were constructed between the maltose-binding protein of Escherichia coli (MalE) and the variable domains (V-domains) of D1.3, a mouse antibody directed against hen lysozyme. Each V-domain was fused with the C- or N-terminus of MalE and expressed in E. coli, either alone or associated with the other V-domain, as a heterodimer (Fv) or as a single-chain fragment (scFv). The hybrids were exported into the bacterial periplasm, purified by affinity chromatography on cross-linked amylose and separated from incomplete products by ion-exchange chromatography. Hybrids between MalE and Fv bound the antigen specifically, with affinities increased up to 10-fold when compared to native D1.3. This strongly suggests that MalE contributed to the binding. The affinities and specificities of the different hybrids, as well as their levels of contamination by incomplete products, depended on their fusion pattern with MalE. Hybrids between MalE and either single V-domain also bound hen lysozyme specifically, which shows that each V-domain can recognize the antigen when fused with MalE. The high affinity of VH-MalE (KD = 3 nM) could be due to both participation of MalE in the binding and a conformational adaptation of the lone V-domain.
Brégégère
F
F
Protein Engineering Group (CNRS URA 1129), Unité de Biochimie Cellulaire, Institut Pasteur, Paris, France.
Schwartz
J
J
Bedouelle
H
H
D018528:ATP-Binding Cassette Transporters; D000818:Animals; D000915:Antibody Affinity; D000937:Antigen-Antibody Reactions; D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics; D001483:Base Sequence; D001666:Binding Sites, Antibody; D002352:Carrier Proteins / Q000737:chemistry* / Q000235:genetics; D004926:Escherichia coli; D029968:Escherichia coli Proteins; D005803:Genes, Immunoglobulin; D005813:Genes, Synthetic; D007143:Immunoglobulin Heavy Chains / Q000737:chemistry / Q000235:genetics / Q000276:immunology; D007147:Immunoglobulin Light Chains / Q000737:chemistry / Q000235:genetics / Q000276:immunology; D007135:Immunoglobulin Variable Region / Q000737:chemistry* / Q000235:genetics; D058619:Maltose-Binding Proteins; D051379:Mice; D008969:Molecular Sequence Data; D009004:Monosaccharide Transport Proteins; D009113:Muramidase / Q000276:immunology; D016297:Mutagenesis, Site-Directed; D033902:Periplasmic Binding Proteins; D015202:Protein Engineering; D011993:Recombinant Fusion Proteins / Q000737:chemistry* / Q000276:immunology / Q000302:isolation & purification
D018528:ATP-Binding Cassette Transporters; D001426:Bacterial Proteins; D002352:Carrier Proteins; D029968:Escherichia coli Proteins; D007143:Immunoglobulin Heavy Chains; D007147:Immunoglobulin Light Chains; D007135:Immunoglobulin Variable Region; C461619:MalE protein, E coli; D058619:Maltose-Binding Proteins; D009004:Monosaccharide Transport Proteins; D033902:Periplasmic Binding Proteins; D011993:Recombinant Fusion Proteins; C461599:maltose transport system, E coli; D009113:Muramidase