Elevated Expression of a Functional Suf Pathway in Escherichia coli BL21(DE3) Enhances Recombinant Production of an Iron-Sulfur Cluster-Containing Protein
Elevated Expression of a Functional Suf Pathway in Escherichia coli BL21(DE3) Enhances Recombinant Production of an Iron-Sulfur Cluster-Containing Protein
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DOI：
10.1128/jb.00494-19
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发表时间：
2020-02-01
影响因子：
3.2
通讯作者：
Antony, Edwin
中科院分区：
文献类型：
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作者：
Corless, Elliot, I;Mettert, Erin L.;Antony, Edwin
含铁-硫[铁-S]簇状蛋白质的结构和光谱分析往往受到重组蛋白的占有率和产率的限制。在这里，我们报道了大肠杆菌BL21(DE3)，一个常规用于过量生产含有[Fe-S]簇蛋白的菌株，具有一个不起作用的Suf途径，这是大肠杆菌[Fe-S]簇的两条生物发生途径之一。我们证实BL21(DE3)和商业上可获得的衍生物携带有缺失，导致SufABCDSE操纵子内SufA和sufB基因的框内融合。我们发现该融合蛋白在细胞中积累，但在[Fe-S]簇的生物发生中不活跃。恢复完整的Suf途径并增强suf操纵子的表达导致含有[4Fe-4S]簇的BCHL蛋白的产量显著增加(类似于3倍)，BCHL蛋白是暗操作原叶绿素氧化还原酶复合体的关键成分。这些结果表明，这种工程化的BL21(DE3)的“SufFeScient”衍生物适合于强化大规模合成含[Fe-S]簇状蛋白，大量重组高产的[Fe-S]簇状蛋白是其深入生化性质所必需的。商业上可获得的大肠杆菌菌株BL21(DE3)及其衍生物具有突变，使负责集群生物发生的两条天然途径(Suf途径)之一的功能失活。突变的纠正，结合提高Suf蛋白水平的序列变化，可以增加[Fe-S]簇酶的产量和簇占有率，促进对这组迷人的蛋白质的生化分析。
Structural and spectroscopic analysis of iron-sulfur [Fe-S] cluster-containing proteins is often limited by the occupancy and yield of recombinantly produced proteins. Here we report that Escherichia coli BL21 (DE3), a strain routinely used to overproduce [Fe-S] cluster-containing proteins, has a nonfunctional Suf pathway, one of two E. coli [Fe-S] cluster biogenesis pathways. We confirmed that BL21(DE3) and commercially available derivatives carry a deletion that results in an in-frame fusion of sufA and sufB genes within the sufABCDSE operon. We show that this fusion protein accumulates in cells but is inactive in [Fe-S] cluster biogenesis. Restoration of an intact Suf pathway combined with enhanced suf operon expression led to a remarkable (similar to 3-fold) increase in the production of the [4Fe-4S] cluster-containing BchL protein, a key component of the dark-operative protochlorophyllide oxidoreductase complex. These results show that this engineered "SufFeScient" derivative of BL21(DE3) is suitable for enhanced large-scale synthesis of an [Fe-S] cluster-containing protein.IMPORTANCE Large quantities of recombinantly overproduced [Fe-S] cluster-containing proteins are necessary for their in-depth biochemical characterization. Commercially available E. coli strain BL21(DE3) and its derivatives have a mutation that inactivates the function of one of the two native pathways (Suf pathway) responsible for cluster biogenesis. Correction of the mutation, combined with sequence changes that elevate Suf protein levels, can increase yield and cluster occupancy of [Fe-S] cluster-containing enzymes, facilitating the biochemical analysis of this fascinating group of proteins.