Fluorescence Enhancement of Fluorescent Unnatural Streptavidin by Binding of a Biotin Analogue with Spacer Tail and Its Application to Biotin Sensing

Hiroaki Shinohara,Xianwei Zhu

doi:10.1155/2014/165369

Abstract

We designed a novel molecular biosensing system for the detection of biotin, an important vitamin by the combination of fluorescent unnatural streptavidin with a commercialized biotin-(AC5)2-hydrazide. A fluorescent unnatural amino acid, BODIPY-FL-aminophenylalanine (BFLAF), was position-specifically incorporated into Trp120 of streptavidin by four-base codon method. Fluorescence of the Trp120BFLAF mutant streptavidin was enhanced by the addition of biotin-(AC5)2-hydrazide with the concentration dependent, whereas fluorescence enhancement was not observed at all by the addition of natural biotin. It was considered that the spacer tail of biotin-(AC5)2-hydrazide may disturb the fluorescence quenching of the Trp120BFLAF by Trp79 and Trp108 of the neighbor subunit. Therefore, biotin sensing was carried out by the competitive binding reaction of biotin-(AC5)2-hydrazide and natural biotin to the fluorescent mutant streptavidin. The fluorescence intensity decreased by increasing free biotin concentration. The result suggested that molecular biosensor for small ligand could be successfully designed by the pair of fluorescent mutant binding protein and ligand analogue.

Highlights

Biotin is a water-soluble B-complex vitamin, known as Vitamin B-7 or Vitamin H
We further discovered that fluorescence of the Trp120BFLAF mutant streptavidin was enhanced when the biotin analogue with a (AC5)2-hydrazide tail was bound to the fluorescent binding protein, whereas natural biotin binding did not induce the fluorescence intensity change
The bands of two mutant streptavidins were confirmed by western blotting, in which the T7-tag of the N-terminal of the mutant streptavidin was recognized with the anti-T7-tag antibody from mouse and the secondary antibody for mouse antibody that was labeled with alkaline phosphatase

Summary

Introduction

Biotin is a water-soluble B-complex vitamin, known as Vitamin B-7 or Vitamin H. The fluorescent mutant streptavidin had enough biotin binding activity as about 70% as a wild type of streptavidin. The biotin binding activity of the wild type and mutant streptavidins was confirmed with dot blot analysis.

Results

Conclusion