A homogeneous assay for relative affinity of binding proteins using a green fluorescent protein tag and membrane disk.
When the association between a ligand immobilized on a membrane disk and a fluorescence-labeled analyte was monitored with a fluorescent microplate reader, the time-dependent increase in fluorescence intensity of the reaction mixture was observed. A novel assay system for the specific interaction based on this phenomenon was designated the homogeneous assay for fluorescence concentrated on membrane (HAFCOM). In this study, streptococcal protein G (SpG) and glycogen-binding subunit R5 of protein phosphatase 1 (PPP1R5) tagged by green fluorescent protein (GFP) were used as the fluorescence-labeled analytes, and the affinity change caused by various amino acid substitutions was measured with HAFCOM. From the site-directed mutagenesis of SpG and PPP1R5, it was clarified that (i) the association rate constant of the Lys454Pro/Glu456Gln mutant of SpG to goat immunoglobulin G was almost equivalent to that of the wild-type but its dissociation rate constant was about 2.7 times that of the wild-type and (ii) the amino acid substitutions of Phe180 in PPP1R5 reduced glycogen-binding by 30-50%. Since HAFCOM using the GFP-tagged analyte requires no special chemicals and instruments, this system can easily and economically assay the specific interaction between target protein and ligand.
Aoki
Takashi
T
Department of Biochemistry, Faculty of Pharmaceutical Sciences, Health Sciences University of Hokkaido, Ishikari-Tobetsu, Hokkaido 061-0293, Japan. aokit@hoku-iryo-u.ac.jp
Kazama
Hitoshi
H
Satoh
Marie
M
Mizuki
Kazuhiro
K
Watabe
Hiroyuki
H
D000818:Animals; D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics; D005470:Fluorometry; D006003:Glycogen / Q000737:chemistry; D049452:Green Fluorescent Proteins / Q000737:chemistry*; D007074:Immunoglobulin G / Q000737:chemistry; D008024:Ligands; D008567:Membranes, Artificial; D051379:Mice; D010749:Phosphoprotein Phosphatases / Q000737:chemistry* / Q000235:genetics; D011145:Polyvinyls / Q000737:chemistry; D011485:Protein Binding; D054645:Protein Phosphatase 1; D005465:Fluorocarbon Polymers
D001426:Bacterial Proteins; D006003:Glycogen; D049452:Green Fluorescent Proteins; D007074:Immunoglobulin G; D008024:Ligands; D008567:Membranes, Artificial; D010749:Phosphoprotein Phosphatases; D011145:Polyvinyls; D054645:Protein Phosphatase 1; C034284:IgG Fc-binding protein, Streptococcus; C024865:polyvinylidene fluoride; D005465:Fluorocarbon Polymers