Biosynthesis of a Fluorescent Cyanobacterial C-phycocyanin Holo-α Subunit in Escherichia coli
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摘要: 为了研究藻蓝蛋白α亚基的生物合成途径,通过构建相容的3种重组质粒pETDuet-cpcA、pCOLADuet-cpcE-cpcF和pACYCDuet-ho1-pcyA,将裂合酶基因cpcE和cpcF、血红素氧化酶基因ho1、藻蓝胆素合成酶基因pcyA和脱辅基藻蓝蛋白α亚基基因cpcA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CpcA-PCB。成功实现了大肠杆菌内藻蓝蛋白α亚基84位半胱氨酸残基与PCB的连接。而在裂合酶基因cpcE和cpcF不转入大肠杆菌的情况下,大肠杆菌内只有0.2%的CpcA-PCB产生。以上研究为进一步在大肠杆菌内合成天然的藻蓝蛋白奠定了基础。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 apoprotein(C-phycocyanin α subunit;cpcA)were expressed on a second plasmid pETDuet-cpcA.Genes for the heterodimeric lyase(cpcE and cpcF)that catalyzes chromophore attachment were expressed on a third plasmid pCOLADuet-cpcE-cpcF.Upon induction,recombinant E.coli used the cellular pool of heme to produce holo-CpcA with spectroscopic properties qualitatively and quantitatively similar to those of the same protein produced endogenously in cyanobacteria.About 0.2% of the apo-CpcA was converted to holo-CpcA-PCB in a similarly engineered E.coli strain that lacks cpcE and cpcF.
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Keywords:
- C-phycocyanin /
- CpcA /
- cpcE /
- cpcF /
- Reconstitution in vivo
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