Combinatorial biosynthesis of Synechocystis PCC6803 phycocyanin holo-α-subunit (CpcA) in Escherichia coli and its activities
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In order to investigate the feasibility for the biosynthetic pathway of CpcA conjugated protein to be reconstituted in Escherichia coli and its antioxidant ability and protective effect on the growth of E. coli, the minimal biosynthetic pathway in cyanobacteria leading from heme to the formation of the cysteinyl residue of phycocyanobilin with deprosthetic CpcA was reconstituted in E. coli using a relatively simple and effective method. When the constructed plasmid pETDuet-6 bearing five genes involved in the biosynthesis of CpcA was transformed into E. coli, the screened transformant acquired a pronounced blue color. Visualization of proteins on SDS-PAGE gel showed a 29 kDa distinct band, corresponding to the theoretically calculated molecular weight of CpcA. Upon exposure to Zn2+ and UV illumination, the CpcA band was fluorescent. Western blot analysis using His-tag monoclonal antibody confirmed the expression of CpcA in the recombinant E. coli. After the optimization of critical medium components by response surface methodology, the recombinant cells produced 22.29 mg/l of CpcA. The recombinant CpcA displayed a strong ability to scavenge three free radicals ·OH, ·DPPH, and O2 − to protect against the oxidation of linoleic acid and to restore the growth of E. coli cells injured by DPPH and H2O2 at a relatively low concentration. These results lay a good foundation for the production and future use of CpcA.
KeywordsPhycocyanin holo-α-subunit conjugated protein Combinatorial biosynthesis Reducing ability Antioxidant activity Cellular oxidative injury
This study was financed by the National Natural Science Foundation of China (No.31171658), the Doctoral Education Foundation of Ministry of Education of China (No.20070353002), and the Youth Talent Program of Zhejiang Gongshang University (No.Q07-09).
Compliance with ethical standards
This article does not contain any studies with human participants or animals performed by any of the authors.
Conflict of interest
The authors declare that they have no competing interests.
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