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A Highly Productive, One-Pot Cell-Free Protein Synthesis Platform Based on Genomically Recoded Escherichia coli
Cell Chemical Biology ( IF 6.762 ) Pub Date : 2019-11-06 , DOI: 10.1016/j.chembiol.2019.10.008
Benjamin J. Des Soye, Vincent R. Gerbasi, Paul M. Thomas, Neil L. Kelleher, Michael C. Jewett

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.
更新日期:2019-11-09

 

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