Regular articleFusion proteins of streptavidin and allophycocyanin alpha subunit for immunofluorescence assay

- Fusion proteins of streptavidin and allophycocyanin alpha subunit were expressed in E. coli.
- The fusion proteins had strong fluorescence and the ability to bind biotin.
- The fusion proteins were used as fluorescent labels in immunofluorescence assay.

Allophycocyanin is generally used as fluorescent label. In this study, the fusion protein (SLA) of core streptavidin from Streptomyces avidinii and allophycocyanin alpha subunit from thermophilic cyanobacterium Thermosynechococcus elongatus BP-1, together with lyase (cpcS) and phycoerythrobilin (PEB) synthesizing enzymes (Ho1 and PebS) or phycocyanobilin (PCB) synthesizing enzymes (Ho1 and PcyA), were co-expressed in Escherichia coli. The fusion proteins were purified by metal affinity chromatography. Zinc staining analysis and spectral analysis showed that the recombinant fusion proteins covalently bound PEB (denoted as SLA-PEB) or PCB (denoted as SLA-PCB). The two proteins were highly fluorescent and were able to binding biotin. α-fetoprotein (AFP), a tumor marker, was used as analyte to evaluate the application of these two fluorescent proteins in sandwich immunofluorescence assay. The measurable ranges of AFP by these proteins were 0.02-50 ng/ml with an average coefficient of variation below 15%. The detection limit for AFP detection by SLA-PEB and SLA-PCB were 0.11 and 0.35 ng/ml, respectively. The detection sensitivities were comparable to that by streptavidin-R-phycoerythrin (SA-PE). Thus, these fusion proteins would be useful fluorescent labels in immunofluorescence assay.