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A Highly Productive, One-Pot Cell-Free Protein Synthesis Platform Based on Genomically Recoded Escherichia coli.
Cell Chemical Biology ( IF 8.6 ) Pub Date : 2019-11-06 , DOI: 10.1016/j.chembiol.2019.10.008
Benjamin J Des Soye 1 , Vincent R Gerbasi 2 , Paul M Thomas 3 , Neil L Kelleher 4 , Michael C Jewett 5
Affiliation  

The site-specific incorporation of non-canonical amino acids (ncAAs) into proteins via amber suppression provides access to novel protein properties, structures, and functions. Historically, poor protein expression yields resulting from release factor 1 (RF1) competition has limited this technology. To address this limitation, we develop a high-yield, one-pot cell-free platform for synthesizing proteins bearing ncAAs based on genomically recoded Escherichia coli lacking RF1. A key feature of this platform is the independence on the addition of purified T7 DNA-directed RNA polymerase (T7RNAP) to catalyze transcription. Extracts derived from our final strain demonstrate high productivity, synthesizing 2.67 ± 0.06 g/L superfolder GFP in batch mode without supplementation of purified T7RNAP. Using an optimized one-pot platform, we demonstrate multi-site incorporation of the ncAA p-acetyl-L-phenylalanine into an elastin-like polypeptide with high accuracy of incorporation and yield. Our work has implications for chemical and synthetic biology.

中文翻译:

基于基因组编码的大肠杆菌的高效,单罐无细胞蛋白质合成平台。

通过琥珀色抑制将非规范性氨基酸(ncAAs)结合到蛋白质中的位点特异性结合提供了获得新的蛋白质特性,结构和功能的途径。从历史上看,由于释放因子1(RF1)竞争而导致的蛋白质表达不佳,限制了该技术。为了解决这个限制,我们开发了一种高产量,一锅无细胞的平台,用于基于基因组编码的缺少RF1的大肠杆菌合成带有ncAA的蛋白质。该平台的关键功能是添加纯化的T7 DNA定向RNA聚合酶(T7RNAP)来催化转录的独立性。来源于我们最终菌株的提取物显示出高生产率,无需补充纯化的T7RNAP即可分批合成2.67±0.06 g / L超级文件夹GFP。使用优化的一锅平台,我们证明了ncAA p-乙酰基-L-苯丙氨酸可以多位点掺入到弹性蛋白样多肽中,并具有很高的掺入和收率准确性。我们的工作对化学和合成生物学具有重要意义。
更新日期:2019-11-09
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