Biosynthesis of a fluorescent cyanobacterial C-phycocyanin holo-α subunit in a heterologous host
Biosynthesis of a fluorescent cyanobacterial C-phycocyanin holo-α subunit in a heterologous host
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DOI：
10.1073/pnas.181340998
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发表时间：
2001-09-11
影响因子：
11.1
通讯作者：
Glazer, AN
中科院分区：
文献类型：
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作者：
Tooley, AJ;Cai, YPA;Glazer, AN
从光合作用的蓝藻(聚球藻)合成荧光全藻胆蛋白亚基的整个过程。PCC6803)在大肠杆菌中重组。蓝藻编码血红素转化为天然发色团3Z-藻蓝胆素所需的酶的基因，即血红素加氧酶1和3Z-藻蓝胆素：铁氧还蛋白氧化还原酶，是在Trp-lac(Trc)启动子的控制下表达的。脱辅基蛋白(C-藻蓝蛋白α亚基)和异源二聚体裂解酶(CPCE和CPCF)的基因从TRC启动子表达到第二个质粒上。在诱导后，重组大肠杆菌利用血红素细胞池产生全息CPCA，其光谱性质在定性和定量上与在蓝藻中产生的相同蛋白质的光谱性质相似。大约三分之一的apo-CPCA转化为holo-CPCA。在缺乏CPCE和CPCF的类似工程的大肠杆菌菌株中，没有发生显著的BLIN加成。这种方法应该可以对藻胆蛋白生物合成中的许多剩余问题进行深入的分析。这些研究还证明了在活细胞中原位产生这些蛋白质的构建物作为荧光蛋白质探针的可行性。
The entire pathway for the synthesis of a fluorescent holophycobiliprotein subunit from a photosynthetic cyanobacterium (Synechocystis sp. PCC6803) was reconstituted in Escherichia coli. Cyanobacterial genes encoding enzymes required for the conversion of heme to the natural chromophore 3Z-phycocyanobilin, namely, heme oxygenase 1 and 3Z-phycocyanobilin:ferredoxin oxidoreductase, were expressed from a plasmid under control of the hybrid trp-lac (trc) promoter. Genes for the apoprotein (C-phycocyanin alpha subunit; cpcA) and the heterodimeric lyase (cpcE and cpcF) that catalyzes chromophore attachment were expressed from the trc promoter on a second plasmid. 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 a third of the apo-CpcA was converted to holo-CpcA. No significant bilin addition took place in a similarly engineered E. coli strain that lacks cpcE and cpcF. This approach should permit incisive analysis of many remaining questions in phycobiliprotein biosynthesis. These studies also demonstrate the feasibility of generating constructs of these proteins in situ for use as fluorescent protein probes in living cells.