Engineering of functional chimeric protein G-Vargula luciferase.
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.
Maeda
Y
Y
Department of Chemistry and Biotechnology, Faculty of Engineering, University of Tokyo, Japan.
Ueda
H
H
Kazami
J
J
Kawano
G
G
Suzuki
E
E
Nagamune
T
T
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
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