A versatile platform for single- and multiple-unnatural amino acid mutagenesis in Escherichia coli.
A versatile platform for single- and multiple-unnatural amino acid mutagenesis in Escherichia coli.
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DOI：
10.1021/bi4000244
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发表时间：
2013-03-12
期刊：
影响因子：
2.9
通讯作者：
Schultz, Peter G.
中科院分区：
文献类型：
--
作者：
Chatterjee, Abhishek;Sun, Sophie B.;Furman, Jennifer L.;Xiao, Han;Schultz, Peter G.
为了将非天然氨基酸（UAA）位点特异性地掺入大肠杆菌中的靶蛋白中，我们使用了抑制质粒，该抑制质粒提供了对感兴趣的UAA特异性的工程抑制tRNA和氨酰-tRNA合成酶（阿尔斯）。进一步提高UAA在E.大肠杆菌中产生了几代抑制质粒。在这里，我们描述了一种新的，高效的抑制质粒，pUltra，窝藏一个单拷贝的tRNA和阿尔斯表达盒，表现出更高的抑制活性比它的前辈。该系统能够有效地将多达三个UAA以高达野生型蛋白表达水平的30%的水平并入相同蛋白中。其独特的复制起点（CloDF 13）和抗生素抗性标记（壮观霉素）允许pUltra与先前报道的pEVOL抑制质粒结合使用，每个质粒编码不同的tRNA/阿尔斯对，以同时将两种不同的UAA插入同一蛋白质中。我们证明了该系统的实用性，有效地将两个生物正交UAA含有酮和叠氮侧链到甾酮异构酶，随后衍生这些氨基酸残基与两个不同的荧光团，能够福斯特共振能量转移相互作用。最后，由于其最小组成，在pUltra中编码两种不同的tRNA/阿尔斯对，从而允许产生能够双重抑制的单个质粒。高抑制效率和携带多个tRNA/阿尔斯对的能力使pUltra成为在大肠杆菌中进行单个和多个UAA诱变的有用系统。杆菌
To site-specifically incorporate an unnatural amino acid (UAA) into target proteins in Escherichia coli, we use a suppressor plasmid that provides an engineered suppressor tRNA and an aminoacyl-tRNA synthetase (aaRS) specific for the UAA of interest. The continuous drive to further improve UAA incorporation efficiency in E. coli has resulted in several generations of suppressor plasmids. Here we describe a new, highly efficient suppressor plasmid, pUltra, harboring a single copy each of the tRNA and aaRS expression cassettes that exhibits higher suppression activity than its predecessors. This system is able to efficiently incorporate up to three UAAs within the same protein at levels up to 30% of the level of wild-type protein expression. Its unique origin of replication (CloDF13) and antibiotic resistance marker (spectinomycin) allow pUltra to be used in conjunction with the previously reported pEVOL suppressor plasmid, each encoding a distinct tRNA/aaRS pair, to simultaneously insert two different UAAs into the same protein. We demonstrate the utility of this system by efficiently incorporating two bio-orthogonal UAAs containing keto and azido side chains into ketosteroid isomerase and subsequently derivatizing these amino acid residues with two distinct fluorophores, capable of Förster resonance energy transfer interaction. Finally, because of its minimal composition, two different tRNA/aaRS pairs were encoded in pUltra, allowing the generation of a single plasmid capable of dual suppression. The high suppression efficiency and the ability to harbor multiple tRNA/aaRS pairs make pUltra a useful system for conducting single- and multiple-UAA mutagenesis in E. coli.
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影响因子：
16.6
作者：
Chen, Peng R.;Groff, Dan;Guo, Jiantao;Ou, Weijia;Cellitti, Susan;Geierstanger, Bernhard H.;Schultz, Peter G.
通讯作者：
Schultz, Peter G.
影响因子：
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作者：
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通讯作者：
Ellington AD
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作者：
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作者：
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通讯作者：
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影响因子：
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作者：
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通讯作者：
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