Cell-free protein synthesis from a release factor 1 deficient Escherichia coli activates efficient and multiple site-specific nonstandard amino acid incorporation.
Cell-free protein synthesis from a release factor 1 deficient Escherichia coli activates efficient and multiple site-specific nonstandard amino acid incorporation.
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DOI：
10.1021/sb400140t
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发表时间：
2014-06-20
影响因子：
4.7
通讯作者：
Jewett, Michael C.
中科院分区：
文献类型：
--
作者：
Hong, Seok Hoon;Ntai, Ioanna;Haimovich, Adrian D.;Kelleher, Neil L.;Isaacs, Farren J.;Jewett, Michael C.
关键词：
将非标准氨基酸（NSAAs）位点特异性地掺入到蛋白质中使得能够产生具有新化学性质、新结构和新功能的生物聚合物、蛋白质和酶。为了实现这一点，琥珀（TAG密码子）抑制已被广泛应用。然而，抑制效率是有限的，由于竞争与翻译终止释放因子1（RF 1），这导致截短的产品。最近，我们构建了一个基因组重新编码的大肠杆菌菌株缺乏RF 1，其中13个琥珀终止密码子的出现已重新分配到同义TAA密码子（rEc. E13.ΔprfA）。在这里，我们评估和特点的无细胞蛋白质合成（CFPS）的粗S30细胞裂解物来源于该菌株。我们观察到190 ± 20 μg/mL的修饰的可溶性超级折叠绿色荧光蛋白（sfGFP）的合成，其含有单个对炔丙氧基-L-苯丙氨酸（pPaF）或对乙酰基-L-苯丙氨酸（pPaF）。与具有RF 1的亲本rEc.E13菌株相比，这导致修饰的sfGFP合成提高超过250%。除了引入单个NSAA之外，我们进一步证明了来自RF 1缺陷型菌株的CFPS在每个sfGFP蛋白的两个和五个位点掺入pPaF的益处。最后，我们将我们的粗S30提取系统与缺乏RF 1的PURE翻译系统进行了比较。我们观察到，我们的基于S30提取物的方法比缺乏RF 1的PURE翻译系统更具成本效益和高产率，以毫克蛋白质产量/美元为基础，大约1000倍。展望未来，使用RF 1缺陷型菌株的提取物为基础的CFPS将有助于蛋白质和生物聚合物的合成与位点特异性纳入NSAAs。
Site-specific incorporation of nonstandard amino acids (NSAAs) into proteins enables the creation of biopolymers, proteins, and enzymes with new chemical properties, new structures, and new functions. To achieve this, amber (TAG codon) suppression has been widely applied. However, the suppression efficiency is limited due to the competition with translation termination by release factor 1 (RF1), which leads to truncated products. Recently, we constructed a genomically recoded Escherichia coli strain lacking RF1 where 13 occurrences of the amber stop codon have been reassigned to the synonymous TAA codon (rEc.E13.ΔprfA). Here, we assessed and characterized cell-free protein synthesis (CFPS) in crude S30 cell lysates derived from this strain. We observed the synthesis of 190 ± 20 μg/mL of modified soluble superfolder green fluorescent protein (sfGFP) containing a single p-propargyloxy-l-phenylalanine (pPaF) or p-acetyl-l-phenylalanine. As compared to the parent rEc.E13 strain with RF1, this results in a modified sfGFP synthesis improvement of more than 250%. Beyond introducing a single NSAA, we further demonstrated benefits of CFPS from the RF1-deficient strains for incorporating pPaF at two- and five-sites per sfGFP protein. Finally, we compared our crude S30 extract system to the PURE translation system lacking RF1. We observed that our S30 extract based approach is more cost-effective and high yielding than the PURE translation system lacking RF1, ∼1000 times on a milligram protein produced/$ basis. Looking forward, using RF1-deficient strains for extract-based CFPS will aid in the synthesis of proteins and biopolymers with site-specifically incorporated NSAAs.
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影响因子：
3.5
作者：
Heinemann IU;Rovner AJ;Aerni HR;Rogulina S;Cheng L;Olds W;Fischer JT;Söll D;Isaacs FJ;Rinehart J
通讯作者：
Rinehart J
影响因子：
64.5
作者：
Nakatogawa, H;Ito, K
通讯作者：
Ito, K
影响因子：
8.4
作者：
Michel-Reydellet, N;Calhoun, K;Swartz, J
通讯作者：
Swartz, J
影响因子：
3.8
作者：
Jewett, MC;Swartz, JR
通讯作者：
Swartz, JR
影响因子：
4
作者：
Johnson, David B. F.;Wang, Chong;Xu, Jianfeng;Schultz, Matthew D.;Schmitz, Robert J.;Ecker, Joseph R.;Wang, Lei
通讯作者：
Wang, Lei