Biosynthesis of a Fluorescent Cyanobacterial Phycoerythrocyanin Holo-α Subunit in Escherichia coli
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摘要: 为了研究藻红蓝蛋白α亚基的生物合成途径,通过构建相容的4种重组质粒pETDuet-pecA、pCOLADuet-pecE、pCDFDuet-pecF和pACYCDuet-ho1-pcyA,将裂合酶基因pecE和pecF、血红素氧化酶基因ho1、藻蓝胆素合成酶基因pcyA和脱辅基藻红蓝蛋白α亚基基因pecA共同转入大肠杆菌BL21(DE3),通过色素蛋白锌电泳和光谱检测表明产生了生物活性的PecA-PCB。结果表明生成的色素藻胆蛋白具有藻红蓝蛋白α-亚基所特有的光谱性质和可逆光致变色性质。而在裂合酶基因pecE和pecF不转入大肠杆菌的情况下,大肠杆菌内只有0.1%的PecA-PCB产生。以上研究对藻胆蛋白生物构建具有重要意义。Abstract: The entire pathway for the synthesis of a fluorescent holophycobiliprotein subunit from a photosynthetic cyanobacterium(Anabaena sp.PCC7120) was reconstituted in Escherichia coli.Cyanobacterial genes for enzymes ho1 and pcyA were expressed from a plasmid pACYCDuet-ho1-pcyA.Genes for the apo-(protein)(Phycoerythrocyanin α subunit;pecA) were expressed on a second plasmid pETDuet-pecA.Genes for the heterodimeric lyase(pecE and pecF) that catalyzes chromophore attachment were expressed on a third plasmid pCOLADuet-pecE and a fourth plasmid pCDFDuet-pecF.Upon induction,recombinant E.coli used the cellular pool of heme to produce holo-PecA with spectroscopic properties qualitatively and quantitatively similar to those of the same protein produced endogenously in cyanobacteria.However,unlike the other biliproteins,isolated PEC shows a pronounced reversible photochemistry,which has been related to the α-subunit(α-PEC).About 0.1% of the apo-PecA was converted to holo-PecA-PCB in a similarly engineered E.coli strain that lacks pecE and pecF.
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Keywords:
- Phycoerythrocyanin /
- PecA /
- pecE /
- pecF /
- Reconstitution in vivo
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