title: Genetically expanded cell-free protein synthesis using endogenous pyrrolysyl orthogonal translation system.
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 accessible platform for UAA incorporation into proteins.
authors.affiliation: Department of Life Sciences and Ilse Katz Institute for Nanoscale Science and Technology, Ben-Gurion University of the Negev, Beer-Sheva, 84105, Israel.
authors.forename: Yonatan
authors.initials: Y
authors.lastname: Chemla
authors.affiliation: Department of Life Sciences and Ilse Katz Institute for Nanoscale Science and Technology, Ben-Gurion University of the Negev, Beer-Sheva, 84105, Israel.
authors.forename: Eden
authors.initials: E
authors.lastname: Ozer
authors.affiliation: Department of Life Sciences and Ilse Katz Institute for Nanoscale Science and Technology, Ben-Gurion University of the Negev, Beer-Sheva, 84105, Israel.
authors.forename: Orr
authors.initials: O
authors.lastname: Schlesinger
authors.affiliation: School of Physics and Astronomy, University of Minnesota, Minneapolis, Minnesota, 55401.
authors.forename: Vincent
authors.initials: V
authors.lastname: Noireaux
authors.affiliation: Department of Life Sciences and Ilse Katz Institute for Nanoscale Science and Technology, Ben-Gurion University of the Negev, Beer-Sheva, 84105, Israel. alfontal@bgu.ac.il.
authors.forename: Lital
authors.initials: L
authors.lastname: Alfonta
mesh_terms: D000596:Amino Acids / Q000378:metabolism; D000604:Amino Acyl-tRNA Synthetases / Q000235:genetics / Q000378:metabolism; D002474:Cell-Free System; D014176:Protein Biosynthesis; D015202:Protein Engineering / Q000379:methods*; D012343:RNA, Transfer / Q000235:genetics / Q000378:metabolism; D012270:Ribosomes / Q000378:metabolism
chemical_list: D000596:Amino Acids; D012343:RNA, Transfer; D000604:Amino Acyl-tRNA Synthetases
keywords: amber suppression; cell-free protein synthesis; genetic code expansion; pyrrolysyl-tRNA synthetase