title: Fluorescent IgG fusion proteins made in E. coli.
abstract: Antibodies are among the most powerful tools in biological and biomedical research and are presently the fastest growing category of new bio-pharmaceutics. The most common format of antibody applied for therapeutic, diagnostic and analytical purposes is the IgG format. For medical applications, recombinant IgGs are made in cultured mammalian cells in a process that is too expensive to be considered for producing antibodies for diagnostic and analytical purposes. Therefore, for such purposes, mouse monoclonal antibodies or polyclonal sera from immunized animals are used. While looking for an easier and more rapid way to prepare full-length IgGs for therapeutic purposes, we recently developed and reported an expression and purification protocol for full-length IgGs, and IgG-based fusion proteins in E. coli, called "Inclonals." By applying the Inclonals technology, we could generate full-length IgGs that are genetically fused to toxins. The aim of the study described herein was to evaluate the possibility of applying the "Inclonals" technology for preparing IgG-fluorophore fusion proteins. We found that IgG fused to the green fluorescent proteins enhanced GFP (EGFP) while maintaining functionality in binding, lost most of its fluorescence during the refolding process. In contrast, we found that green fluorescent Superfolder GFP (SFGFP)-fused IgG and red fluorescent mCherry-fused IgG were functional in antigen binding and maintained fluorescence intensity. In addition, we found that we can link several SFGFPs in tandem to each IgG, with fluorescence intensity increasing accordingly. Fluorescent IgGs made in E. coli may become attractive alternatives to monoclonal or polyclonal fluorescent antibodies derived from animals.
authors.affiliation: Department of Molecular Microbiology and Biotechnology, The George S. Wise Faculty of Life Sciences, Ramat Aviv, Israel.
authors.forename: Yael
authors.initials: Y
authors.lastname: Luria
authors.forename: Dina
authors.initials: D
authors.lastname: Raichlin
authors.forename: Itai
authors.initials: I
authors.lastname: Benhar
mesh_terms: D000818:Animals; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism*; D005240:Feasibility Studies; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D007074:Immunoglobulin G / Q000235:genetics / Q000378:metabolism*; D008164:Luminescent Proteins / Q000235:genetics / Q000378:metabolism*; D051379:Mice; D015335:Molecular Probes / Q000378:metabolism; D011485:Protein Binding / Q000235:genetics; D017510:Protein Folding; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism*; D000097573:Red Fluorescent Protein
chemical_list: D049452:Green Fluorescent Proteins; D007074:Immunoglobulin G; D008164:Luminescent Proteins; D015335:Molecular Probes; D011993:Recombinant Fusion Proteins; D000097573:Red Fluorescent Protein