Biosynthesis of a Phycocyanin Beta Subunit with Two Noncognate Chromophores in Escherichia coli
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Biosynthesis of a Phycocyanin Beta Subunit with Two Noncognate Chromophores in Escherichia coli Huaxin Chen 1,2,3
4
& Caiyun Zheng & Peng Jiang
1,2,3
& Gengsheng Ji
4
Received: 29 September 2019 / Accepted: 5 December 2019/ # Springer Science+Business Media, LLC, part of Springer Nature 2019
Abstract
Recombinant phycobiliprotein can be used as fluorescent label in immunofluorescence assay. In this study, pathway for phycocyanin beta subunit (CpcB) carrying noncognate chromophore phycoerythrobilin (PEB) and phycourobilin (PUB) was constructed in Escherichia coli. Lyase CpcS and CpcT could catalyze attachment of PEB to Cys84CpcB and Cys155-CpcB, respectively. However, PEB was attached only to Cys84-CpcB when both CpcS and CpcT were present in E. coli. A dual plasmid expression system was used to control the expression of lyases and the attachment order of PEB to CpcB. The production of PEB-Cys155-CpcB was achieved by L-arabinose-induced expression of CpcS, CpcB, Ho1, and PebS, and then the attachment of PEB to Cys84-CpcB was achieved by IPTG-induced expression of CpcS. The doubly chromophorylated CpcB absorbed light maximally at 497.5 nm and 557.0 nm and fluoresced maximally at 507.5 nm and 566.5 nm. An amount of light energy absorbed by PUB-Cys155-CpcB is transferred to PEB-Cys84-CpcB in doubly chromophorylated CpcB, conferring a large stokes shift of 69 nm for this fluorescent protein. There are interactions between chromophores of CpcB which possibly together with the help of lyases lead to isomerization of PEB-Cys155-CpcB to PUB-Cys155-CpcB. Keywords Phycobiliprotein . Phycocyanin . Lyase . Fluorescence . Chromophore . E. coli
* Huaxin Chen [email protected]
1
Key Laboratory of Experimental Marine Biology, Institute of Oceanology Chinese Academy of Sciences, Qingdao 266071, China
2
Laboratory for Marine Biology and Biotechnology, Qingdao National Laboratory for Marine Science and Technology, Qingdao 266071, China
3
Center for Ocean Mega-Science, Chinese Academy of Sciences, Qingdao 266071, China
4
College of Biotechnology Sericultural Research Institute, Jiangsu University of Science and Technology, Jiangsu, China
Applied Biochemistry and Biotechnology
Introduction Phycobiliproteins are photosynthetic light-harvesting antennae found in cyanobacteria, red algae, and cryptomonds. Among the four phycobiliproteins, viz., phycocyanin (PC), phycoerythrin (PE), allophycocyanin (APC), and phycerythrocyanin (PEC), the most common is PC [1]. The phycobiliproteins covalently bind linear tetrapyrrole phycobilins and absorb visible light ranging from 490 nm to 700 nm. As highly fluorescent proteins, the phycobiliproteins exhibit exceptional spectral properties, such as high extinction coefficient and fluorescence quantum yield. They have been widely used as fluorescent labels to probe biological events [2]. Commercial phycobiliproteins are extracted and purified from cyanobacteria [3]. In recent decades, the pathway for phycobiliprotein biosynthesis has been elucidated [4–7]. This progress makes it possible
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