A Highly Productive, One-Pot Cell-Free Protein Synthesis Platform Based on Genomically Recoded Escherichia coli
A Highly Productive, One-Pot Cell-Free Protein Synthesis Platform Based on Genomically Recoded Escherichia coli
复制标题
DOI：
10.1016/j.chembiol.2019.10.008
复制
发表时间：
2019-12-19
影响因子：
8.6
通讯作者：
Jewett, Michael C.
中科院分区：
文献类型：
--
作者：
Des Soye, Benjamin J.;Gerbasi, Vincent R.;Jewett, Michael C.
非规范氨基酸（ncAAs）通过琥珀抑制进入蛋白质的位点特异性结合提供了新的蛋白质特性、结构和功能。从历史上看，释放因子1 （RF1）竞争导致的蛋白表达量低限制了这项技术。为了解决这一限制，我们开发了一种高产、一锅无细胞平台，用于合成基于基因组编码的缺乏RF1的大肠杆菌的含ncaa蛋白质。该平台的一个关键特点是不依赖于添加纯化的T7 dna定向RNA聚合酶（T7RNAP）来催化转录。最终菌株的提取物显示出较高的生产效率，在不添加纯化的T7RNAP的情况下，批量模式下合成了2.67 +/- 0.06 g/L的超级文件夹GFP。利用优化的单锅平台，我们证明了ncAA p-乙酰-l -苯丙氨酸与弹性蛋白样多肽的多位点结合，结合精度高，产率高。我们的工作对化学和合成生物学有影响。
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.