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.

Huaxin Chen, Peng Jiang

Journal: Journal of bioscience and bioengineering 2019;126(6):778-782

PMID: 30401453

Full Text on Elsevier Science View on PubMed

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.

Address: CAS Key Laboratory of Experimental Marine Biology, Institute of Oceanology, Chinese Academy of Sciences, Qingdao 266071, China; Laboratory for Marine Biology and Biotechnology, Qingdao National Laboratory for Marine Science and Technology, Qingdao 266071, China; Center for Ocean Mega-Science, Chinese Academy of Sciences, Qingdao 266071, China. Electronic address: [email protected].; CAS Key Laboratory of Experimental Marine Biology, Institute of Oceanology, Chinese Academy of Sciences, Qingdao 266071, China; Laboratory for Marine Biology and Biotechnology, Qingdao National Laboratory for Marine Science and Technology, Qingdao 266071, China; Center for Ocean Mega-Science, Chinese Academy of Sciences, Qingdao 266071, China.

DOI: 10.1016/j.jbiosc.2018.06.004

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MeSH Terms: Chromatography, Affinity, Escherichia coli, Fluorescent Antibody Technique, Fluorescent Dyes, Lyases, Phycobilins, Phycocyanin, Phycoerythrin, Protein Subunits, Recombinant Fusion Proteins, Streptavidin