Construction of a fusion protein between protein A and green fluorescent protein and its application to western blotting.
Aequorea green fluorescent protein (GFP) and protein A were fused and expressed in Escherichia coli. The fluorescent native fusion protein (PA-GFP) migrated at 47 kDa in SDS-PAGE. However, the non-fluorescent denatured PA-GFP migrated at 57 kDa which corresponds to the theoretical molecular mass. Although the reason(s) for this mobility shift between fluorescent and non-fluorescent molecules remains unclear, the small ring structure within the native molecules may affect their mobility. The cell extract, prepared from an E. coli strain producing PA-GFP, was used in Western and dot blots. The sensitivity and specificity of the PA-GFP detection were sufficient for rapid and easy screening.
Aoki
T
T
Department of Biochemistry, Faculty of Pharmaceutical Sciences, Health Sciences University of Hokkaido, Japan.
Takahashi
Y
Y
Koch
K S
KS
Leffert
H L
HL
Watabe
H
H
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D015153:Blotting, Western; D018076:DNA, Complementary / Q000235:genetics; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000378:metabolism; D005456:Fluorescent Dyes / Q000032:analysis*; D049452:Green Fluorescent Proteins; D008164:Luminescent Proteins / Q000032:analysis* / Q000096:biosynthesis / Q000235:genetics; D008969:Molecular Sequence Data; D010751:Phosphopyruvate Hydratase / Q000276:immunology; D016133:Polymerase Chain Reaction; D011817:Rabbits; D011993:Recombinant Fusion Proteins / Q000032:analysis* / Q000096:biosynthesis / Q000737:chemistry; D012680:Sensitivity and Specificity; D013205:Staphylococcal Protein A / Q000032:analysis / Q000096:biosynthesis / Q000235:genetics*
D018076:DNA, Complementary; D005456:Fluorescent Dyes; D008164:Luminescent Proteins; D011993:Recombinant Fusion Proteins; D013205:Staphylococcal Protein A; D049452:Green Fluorescent Proteins; D010751:Phosphopyruvate Hydratase