title: Engineering of functional chimeric protein G-Vargula luciferase.
abstract: Luciferase of Vargula hilgendorfli is infinitely stable at room temperature in dried state, and its light-emitting reaction is very simple. These unique characteristics of Vargula luciferase have prompted us to engineer chimeric protein, the other moiety chosen for conjugation being streptococcal protein G. A single domain of protein G which binds to IgG of a wide range of species was fused at the N-terminal region of Vargula luciferase. Unexpectedly, we found that the chimeric protein expressed in mammalian COS-1 cells had no IgG-binding ability, probably due to some sort of interaction between the two moieties or some conformational preferences of the IgG-binding domain of protein G when fused to Vargula luciferase. Here we report how we regained the IgG binding of protein G, by the intervention of three alpha-helices of protein A between protein G and luciferase. To our knowledge, the new chimeric protein provides the first reported model of this kind.
authors.affiliation: Department of Chemistry and Biotechnology, Faculty of Engineering, University of Tokyo, Japan.
authors.forename: Y
authors.initials: Y
authors.lastname: Maeda
authors.forename: H
authors.initials: H
authors.lastname: Ueda
authors.forename: J
authors.initials: J
authors.lastname: Kazami
authors.forename: G
authors.initials: G
authors.lastname: Kawano
authors.forename: E
authors.initials: E
authors.lastname: Suzuki
authors.forename: T
authors.initials: T
authors.lastname: Nagamune
mesh_terms: D000818:Animals; D019556:COS Cells; D003445:Crustacea / Q000201:enzymology*; D004274:DNA, Recombinant; D005822:Genetic Vectors; D007141:Immunoglobulin Fc Fragments / Q000378:metabolism; D007074:Immunoglobulin G / Q000378:metabolism; D008156:Luciferases / Q000096:biosynthesis / Q000235:genetics* / Q000378:metabolism; D009419:Nerve Tissue Proteins / Q000096:biosynthesis / Q000235:genetics* / Q000378:metabolism; D011485:Protein Binding; D015202:Protein Engineering / Q000379:methods; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000235:genetics* / Q000378:metabolism; D013205:Staphylococcal Protein A / Q000235:genetics / Q000378:metabolism; D013291:Streptococcus; D014162:Transfection
chemical_list: D004274:DNA, Recombinant; C029152:G-substrate; D007141:Immunoglobulin Fc Fragments; D007074:Immunoglobulin G; D009419:Nerve Tissue Proteins; D011993:Recombinant Fusion Proteins; D013205:Staphylococcal Protein A; D008156:Luciferases