Combinational biosynthesis and characterization of fusion proteins with tandem repeats of allophycocyanin holo-α subunits, and their application as bright fluorescent labels for immunofluorescence assay

Abstract

Fusion protein of streptavidin and allophycocyanin holo-α subunit (ApcA) is fluorescent and is able to bind biotin. This fusion protein (SLA) can be used as fluorescent label in immunofluorescence assay. In this study, one or two repeats of ApcA were fused to the protein SLA, with the aim to improve its brightness. The fusion proteins SLA2 (two repeats of ApcA) and SLA3 (three repeats of ApcA), together with lyase (cpcS) and phycoerythrobilin synthesizing enzymes (Ho1 and PebS), were co-expressed in Escherichia coli. These fusion proteins were purified by affinity chromatography. While SLA2 and SLA3 shared similar absorbance spectra, fluorescence spectra and biotin-binding activities with SLA, their brightness were much higher than that of SLA. When used as fluorescent labels in immunofluorescence assay, SLA2 and SLA3 showed higher detection sensitivity than SLA. These results suggested that SLA2 and SLA3 were the preferable fluorescent labels in immunofluorescence assays.

Keywords: Allophycocyanin; Fluorescence; Fusion protein; Immunofluorescence assay; Streptavidin.