Simple, functional, inexpensive cell extract for in vitro prototyping of proteins with disulfide bonds
Simple, functional, inexpensive cell extract for in vitro prototyping of proteins with disulfide bonds
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DOI：
10.1016/j.bej.2020.107790
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发表时间：
2020-12-15
影响因子：
3.9
通讯作者：
Reuel, Nigel F.
中科院分区：
文献类型：
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作者：
Dopp, Jared L.;Reuel, Nigel F.
体外表达大肠杆菌提取物的蛋白质是一种有用的方法，用于原型和生产细胞毒性或非天然产品。然而，具有多个二硫键的蛋白质需要定制提取物，迄今为止，需要小心地添加外源异构酶或使用昂贵的商业试剂盒。这种成本和复杂性目前限制了一些希望快速构建具有二硫键的蛋白质原型的团队。在这里，我们提出了一个简单的解决方案，不需要添加补充酶。我们使用市售的SHuffle T7 Express lysY大肠杆菌菌株，该菌株同时表达T7 RNAP和DsbC异构酶。我们通过实验确定最佳生长条件（IPTG诱导和收获时间），以平衡总体生产力和二硫键形成的效率，使用含有五个二硫键的荧光素酶（来自Gaussia princeps）作为我们的报告蛋白。我们还通过筛选四种推测的荧光素酶对十种荧光素类似物的活性来证明快速分型的能力。为了显示提取物的广泛适用性，我们还从最小的基因模板中表达了含有>= 3二硫键的其他三种酶（hevamine， endochitinase A和periplasmic AppA），这些基因模板经过滚圈扩增并通过活性测定证实。
In vitro expression of proteins from E. coli extract is a useful method for prototyping and production of cytotoxic or unnatural products. However, proteins that have multiple disulfide bonds require custom extract that, to date, requires careful addition of exogenous isomerase enzymes or the use of expensive commercial kits. This cost and complexity currently limit access to some groups who wish to rapidly prototype proteins with disulfide bonds. Herein, we present a simple solution that does not require addition of supplemental enzymes. We use a commercially available SHuffle T7 Express lysY strain of E. coli that expresses both T7 RNAP and DsbC isomerase enzymes. We experimentally determine optimal growth conditions (IPTG induction and harvest times) to balance overall productivity and efficiency of disulfide bond formation using a luciferase (from Gaussia princeps) that contains five disulfide bonds as our reporter protein. We also demonstrate the ability for rapid pmtotyping by screening the activity of four putative luciferases against ten luciferin analogues. To display the broad applicability of the extract, three other enzymes containing >= 3 disulfide bonds (hevamine, endochitinase A, and periplasmic AppA) were also expressed from minimal genetic templates that had undergone rolling circle amplification and confirmed via activity assays.