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Combinational Biosynthesis of a Fluorescent Cyanobacterial Holo-α-Phycocyanin in Escherichia coli by Using One Expression Vector

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Abstract

The phycobiliproteins (PBSs) are large pigment proteins found in certain algae that play a central role in harvesting light energy for photosynthesis. Phycocyanin (PC) is one type of PBSs that gains increasing attention owing to its various biological and pharmacological properties. In this paper, an expression vector containing five essential genes in charge of biosynthesis of cyanobacterial C-phycocyanin (C-PC) holo-α subunit (holo-CpcA) was successfully constructed resulting in over-expression of a fluorescent holo-CpcA in E. coli BL21. The vector harbored two cassettes: one cassette carried genes hox1 and pcyA required for conversion of heme to phycocyanobilin (PCB), and the other cassette carried cpcA encoding CpcA along with cpcE and cpcF both of which were necessary and sufficient for the correct addition of PCB to CpcA. The vector system contained a His-tag for protein purification. The purified protein showed correct molecular weight on SDS-PAGE gel and emitted orange fluorescence by UV excitation. The maximum peak of absorbance spectrum was at 623 nm, and the maximum peak of fluorescence emission and excitation were at 648 and 633 nm, respectively, which were similar to those of native C-PC. This study provides an efficient method for large-scale production of the fluorescent holo-CpcA in biotechnological applications.

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Abbreviations

PBS:

phycobiliproteins

CpcA:

α subunit of C-phycocyanin

CpcE and CpcF:

subunits of the heterodimeric phycocyanin α-subunit phycocyanobilin lyase

Hox1:

heme oxygenase 1

HT:

His-tag

PCB:

phycocyanobilin

PcyA:

3Z-phycocyanobilin: ferredoxin oxidoreductase

Sp:

spectinomycin

Km:

kanamycin

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Acknowledgement

This work was supported by grants from Key Innovative Project of Chinese Academy of Sciences (KZCX-3-SW-215).

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Authors and Affiliations

  1. College of Marine Life Science, Faculty of Life Science and Technology, Ocean University of China, 266003, Qingdao, People’s Republic of China

    Xiangyu Guan & Xuexi Tang

  2. Institute of Oceanology, Chinese Academy of Sciences, 266071, Qingdao, People’s Republic of China

    Song Qin, Fangqing Zhao, Baosheng Ge & Fuchao Li

  3. Qingdao University of Science & Technology, 266006, Qingdao, People’s Republic of China

    Zhongliang Su

Authors
  1. Xiangyu Guan
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  2. Song Qin
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  3. Zhongliang Su
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  4. Fangqing Zhao
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  5. Baosheng Ge
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  6. Fuchao Li
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  7. Xuexi Tang
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Corresponding author

Correspondence to Xuexi Tang.

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Guan and Qin contributed equally to this study.

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Guan, X., Qin, S., Su, Z. et al. Combinational Biosynthesis of a Fluorescent Cyanobacterial Holo-α-Phycocyanin in Escherichia coli by Using One Expression Vector. Appl Biochem Biotechnol 142, 52–59 (2007). https://doi.org/10.1007/s12010-007-8000-7

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  • DOI: https://doi.org/10.1007/s12010-007-8000-7

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