Fluorescence Enhancement of Fluorescent Unnatural Streptavidin by Binding of a Biotin Analogue with Spacer Tail and Its Application to Biotin Sensing
(a) The result of western blotting analysis for wild type, Tyr83BFLAF and Trp120BFLAF mutant streptavidins. BFLAF was position-specifically incorporated into streptavidin by using a CGGG four-base codon. The bands of the wild and mutant streptavidins were observed around 20 kDa. (b) The result of dot blotting. The biotin binding activities of these fluorescent mutant streptavidin were evaluated by a dot blot analysis using an alkaline phosphatase-labeled biotin. The protein amounts of Tyr83BFLAF and Trp120BFLAF mutant streptavidins blotted on the membrane were 15.2% and 14.4% of that of wild type streptavidin.