Detection of Incorporation of p-Coumaric Acid into Photoactive Yellow Protein Variants in Vivo.
Detection of Incorporation of p-Coumaric Acid into Photoactive Yellow Protein Variants in Vivo.
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DOI：
10.1021/acs.biochem.9b00279
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发表时间：
2019-06
期刊：
影响因子：
2.9
通讯作者：
Katherine E. Brechun;Danlin Zhen;A. Jaikaran;V. Borisenko;M. Kumauchi;W. Hoff;K. Arndt;G. Woolley
中科院分区：
文献类型：
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作者：
Katherine E. Brechun;Danlin Zhen;A. Jaikaran;V. Borisenko;M. Kumauchi;W. Hoff;K. Arndt;G. Woolley
我们报道了光活性黄色蛋白(PYP)-蓝色荧光蛋白（mTagBFP）融合构建物的设计和表征，该融合构建物允许直接检测PYP结构域的重构和功能。这些结构允许通过酪氨酸解氨酶和对香豆酸辅酶a连接酶（pCL或4CL）的作用，对香豆酸基PYP发色团的酶促生产共表达系统进行体内测试。研究人员发现，不同的4CL酶可以重组PYP，包括拟南芥中的4CL，在最佳条件下可以产生~ 100%的全PYP蛋白。mTagBFP融合构建还可以快速分析突变对PYP光循环的影响。我们使用这种mTagBFP融合策略，通过生物合成的发色团在大肠杆菌中展示了几种基于pypp的光遗传工具的体内重组，这是在不同宿主中使用这些光遗传工具的重要一步。
We report the design and characterization of photoactive yellow protein (PYP)-blue fluorescent protein (mTagBFP) fusion constructs that permit the direct assay of reconstitution and function of the PYP domain. These constructs allow for in vivo testing of co-expression systems for enzymatic production of the p-coumaric acid-based PYP chromophore, via the action of tyrosine ammonia lyase and p-coumaroyl-CoA ligase (pCL or 4CL). We find that different 4CL enzymes can function to reconstitute PYP, including 4CL from Arabidopsis thaliana that can produce ∼100% holo-PYP protein under optimal conditions. mTagBFP fusion constructs additionally enable rapid analysis of effects of mutations on PYP photocycles. We use this mTagBFP fusion strategy to demonstrate in vivo reconstitution of several PYP-based optogenetic tools in Escherichia coli via a biosynthesized chromophore, an important step for the use of these optogenetic tools in vivo in diverse hosts.