Genetically expanded cell-free protein synthesis using endogenous pyrrolysyl orthogonal translation system

Biotechnology and Bioengineering
Yonatan ChemlaLital Alfonta

Abstract

Cell-free protein synthesis offers a facile and rapid method for synthesizing, monitoring, analyzing, and purifying proteins from a DNA template. At the same time, genetic code expansion methods are gaining attention due to their ability to site-specifically incorporate unnatural amino acids (UAAs) into proteins via ribosomal translation. These systems are based on the exogenous addition of an orthogonal translation system (OTS), comprising an orthogonal tRNA, and orthogonal aminoacyl tRNA synthetase (aaRS), to the cell-free reaction mixture. However, these components are unstable and their preparation is labor-intensive, hence introducing a major challenge to the system. Here, we report on an approach that significantly reduces the complexity, effort and time needed to express UAA-containing proteins while increasing stability and realizing maximal suppression efficiency. We demonstrate an endogenously introduced orthogonal pair that enables the use of the valuable yet insoluble pyrrolysyl-tRNA synthetase in a cell-free system, thereby expanding the genetic repertoire that can be utilized in vitro and enabling new possibilities for bioengineering. With the high stability and efficiency of our system, we offer an improved and a...Continue Reading

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Citations

Jan 29, 2016·ACS Synthetic Biology·Jonathan GaramellaVincent Noireaux
Nov 25, 2016·Biotechnology and Bioengineering·Eden OzerLital Alfonta
Apr 11, 2017·Biochemistry·Yonatan ChemlaLital Alfonta
May 20, 2017·Chemical Communications : Chem Comm·Sagardip MajumderAllen P Liu
Mar 11, 2020·Annual Review of Biomedical Engineering·Vincent Noireaux, Allen P Liu
May 7, 2016·Chembiochem : a European Journal of Chemical Biology·Ki Baek LeeTaek Jin Kang
Oct 16, 2016·Cold Spring Harbor Perspectives in Biology·Jessica G PerezMichael C Jewett
Nov 30, 2019·Nucleic Acids Research·Michael J HammerlingMichael C Jewett
Nov 5, 2019·Frontiers in Bioengineering and Biotechnology·Khushal KhambhatiVijai Singh
May 11, 2021·ACS Synthetic Biology·Damian Van Raad, Thomas Huber
Oct 31, 2019·Bioconjugate Chemistry·Ofir RegevEyal Gur
Feb 15, 2019·ACS Synthetic Biology·Yoshiki MoriizumiHiroyuki Noji

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