Specific ligand binding attributable to individual epitopes of gonococcal transferrin binding protein A.
The gonococcal transferrin receptor complex comprises two iron-regulated proteins, TbpA and TbpB. TbpA is essential for transferrin-iron uptake and is a TonB-dependent integral outer membrane protein. TbpB is thought to increase the efficiency of iron uptake from transferrin and is lipid modified and surface exposed. To evaluate the structure-function relationships in one of the components of the receptor, TbpA, we created constructs that fused individual putative loops of TbpA with amino-terminal affinity tags. The recombinant proteins were then overexpressed in Escherichia coli, and the fusions were recovered predominately from inclusion bodies. Inclusion body proteins were solubilized, and the epitope fusions were renatured by slow dialysis. To assess transferrin binding capabilities, the constructs were tested in a solid-phase dot blot assay followed by confirmatory quantitative chemiluminescent enzyme-linked immunosorbent assays. The constructs with only loop 5 and with loops 4 and 5 demonstrated dose-dependent specific ligand binding in spite of being out of the context of the intact receptor. The immunogenicities of individual TbpA-specific epitopes were investigated by generating rabbit polyclonal antisera against the fusion proteins. Most of the fusion proteins were immunogenic under these conditions, and the resulting sera recognized full-length TbpA in immunoblots. These results suggest that individual epitopes of TbpA are both immunogenic and functional with respect to ligand binding capabilities, and the vaccine implications of these findings are discussed.
Masri
Heather P
HP
Department of Microbiology and Immunology, Medical College of Virginia Campus, Virginia Commonwealth University, Richmond, Virginia 23298-0678, USA.
Cornelissen
Cynthia Nau
CN
D000818:Animals; D001425:Bacterial Outer Membrane Proteins / Q000235:genetics / Q000276:immunology* / Q000378:metabolism; D001665:Binding Sites; D002352:Carrier Proteins / Q000235:genetics / Q000276:immunology* / Q000378:metabolism; D018985:Epitopes, B-Lymphocyte / Q000235:genetics / Q000276:immunology* / Q000378:metabolism; D005260:Female; D015870:Gene Expression; D006801:Humans; D033862:Iron-Binding Proteins; D008024:Ligands; D008163:Luminescent Measurements; D009344:Neisseria gonorrhoeae / Q000235:genetics / Q000276:immunology* / Q000378:metabolism; D011485:Protein Binding; D011817:Rabbits; D011990:Receptors, Transferrin / Q000235:genetics / Q000276:immunology* / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000276:immunology / Q000378:metabolism; D012685:Sepharose; D014168:Transferrin / Q000378:metabolism*; D033863:Transferrin-Binding Proteins
D001425:Bacterial Outer Membrane Proteins; D002352:Carrier Proteins; D018985:Epitopes, B-Lymphocyte; D033862:Iron-Binding Proteins; D008024:Ligands; D011990:Receptors, Transferrin; D011993:Recombinant Fusion Proteins; D014168:Transferrin; D033863:Transferrin-Binding Proteins; D012685:Sepharose