title: Combinatorial biosynthesis of Synechocystis PCC6803 phycocyanin holo-α-subunit (CpcA) in Escherichia coli and its activities.
abstract: 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 Zn(2+) 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.
authors: College of Food Science and Biotechnology, Zhejiang Gongshang University, 149 Jiaogong Road, Hangzhou, 310035, Zhejiang Province, People's Republic of China. yup9202@hotmail.com.
authors: Ping
authors: P
authors: Yu
authors: College of Food Science and Biotechnology, Zhejiang Gongshang University, 149 Jiaogong Road, Hangzhou, 310035, Zhejiang Province, People's Republic of China.
authors: Peng
authors: P
authors: Li
authors: College of Food Science and Biotechnology, Zhejiang Gongshang University, 149 Jiaogong Road, Hangzhou, 310035, Zhejiang Province, People's Republic of China.
authors: Xingge
authors: X
authors: Chen
authors: College of Food Science and Biotechnology, Zhejiang Gongshang University, 149 Jiaogong Road, Hangzhou, 310035, Zhejiang Province, People's Republic of China.
authors: Xiaoyin
authors: X
authors: Chao
mesh_terms: D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism; D053898:Biosynthetic Pathways; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000235:genetics* / Q000254:growth & development / Q000378:metabolism*; D005609:Free Radicals / Q000378:metabolism; D005822:Genetic Vectors; D006861:Hydrogen Peroxide / Q000378:metabolism; D052980:Phycobilins / Q000235:genetics / Q000378:metabolism; D010798:Phycocyanin / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D010957:Plasmids; D011994:Recombinant Proteins / Q000096:biosynthesis; D046939:Synechocystis / Q000235:genetics* / Q000378:metabolism; D014169:Transformation, Bacterial
chemical_list: D001426:Bacterial Proteins; D005609:Free Radicals; D052980:Phycobilins; D011994:Recombinant Proteins; D010798:Phycocyanin; C011069:phycocyanobilin; D006861:Hydrogen Peroxide
keywords: Antioxidant activity; Cellular oxidative injury; Combinatorial biosynthesis; Phycocyanin holo-α-subunit conjugated protein; Reducing ability