Cloning and expression of Allophycocyanin gene from Gracilariopsis lemaneiformis and studying the binding sites of phyco
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23RD INTERNATIONAL SEAWEED SYMPOSIUM, JEJU
Cloning and expression of Allophycocyanin gene from Gracilariopsis lemaneiformis and studying the binding sites of phycocyanobilin on its α and β subunits Yalin Guo 1 & Xiaonan Zang 1 & Xuexue Cao 1 & Feng Zhang 1 & Deguang Sun 1 & Menghui Shang 1 & Rui Li 1 & Zhaxi Yangzong 1 & Xuehong Wei 1 & Xuecheng Zhang 1 Received: 29 June 2019 / Revised and accepted: 9 March 2020 # Springer Nature B.V. 2020
Abstract Allophycocyanin (APC) is a pigment-protein with optical activity in the core of phycobilisomes of cyanobacteria and red algae. Its wide application prospects make it necessary to carry out the recombinant expression of allophycocyanin with optical activity. In this study, apcA and apcB genes encoding two subunits of allophycocyanin were cloned from Gracilariopsis lemaneiformis. Then apcA and apcB genes expressed in Escherichia coli to obtain allophycocyanin. In order to investigate the active sites of allophycocyanin to bind with phycocyanobilin, the Cys-81 and Cys-138 of α subunit and Cys-81 and Cys-157 of β subunit were mutated. SDS-PAGE and Western blotting results verified the expression of allophycocyanin. The fluorescence emission spectra showed the characteristic fluorescence peak of allophycocyanin, which indicated that the recombinant allophycocyanin had optical activity. The recombinant strains with chromophore lyases—CpcU and CpcS—had the higher fluorescence emission peak, indicating that chromophore lyases would catalyze the combination of phycocyanobilin and apo-allophycocyanin more effectively. The binding sites were Cys-81 and Cys138 of allophycocyanin α subunit, and Cys-81 and Cys-157 of allophycocyanin β subunit. The catalytic effect of CpeT was not obvious. This research provides an experimental foundation for understanding the synthesis mechanism of optically active allophycocyanin in G. lemaneiformis. Keywords Gracilariopsis lemaneiformis . Rhodophyta . Allophycocyanin . Expression . Fluorescence
Introduction The phycobilisomes are highly ordered supramolecular complexes that can absorb and transmit light energy in cyanobacteria and red algae. At present, the resolution of the overall structure analysis of phycobilisome has reached 3.5 Å by single-particle cryo-electron microscopy (Zhang et al. 2017). The phycobilisomes consist of heterodimeric phycobiliproteins and linker polypeptides such that the accurate assembly of phycobilisome and transmission of light energy to photosystem II are optimized (Arteni et al. 2009). According to the different absorption spectra, the known phycobiliproteins are mainly
* Xiaonan Zang [email protected] 1
Key Laboratory of Marine Genetics and Breeding, Ocean University of China, Ministry of Education, Qingdao 266003, Shandong, China
composed of four types, namely phycoerythrin (PE), λmax = 540 to 570 nm; phycoerythrocyanin (PEC), λmax = 567 nm; phycocyanin (PC), λmax = 615 to 640 nm, and allophycocyanin (APC), λmax = 650 to 655 nm. Phycobiliprotein is usually composed of α subunit and β subunit (γ subunit is also
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