RESUMO
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.
Assuntos
Proteínas de Bactérias/química , Proteínas de Fluorescência Verde/química , Fosfoproteínas Fosfatases/química , Ligação Proteica , Animais , Proteínas de Bactérias/genética , Fluorometria , Glicogênio/química , Imunoglobulina G/química , Ligantes , Membranas Artificiais , Camundongos , Fosfoproteínas Fosfatases/genética , Polivinil/química , Proteína Fosfatase 1RESUMO
Affinity electrophoresis (AEP) using green fluorescent protein (GFP) was studied. We constructed a fusion protein that linked S147PGFP and IgG binding domain C from streptococcal protein G (GFP-SpGC). The affinity of GFP-SpGC for mouse IgG1 was measured. The AEP using GFP does not require a staining step after electrophoresis, and was successful with a non-purified sample. Therefore, this method is simple and useful for measuring many samples such as those used in mutational studies.
