title: In vitro suppression of two different stop codons.
abstract: Proteins play a crucial role in all living organisms, with the 20 natural amino acids as their building blocks. Unnatural amino acids are synthetic derivatives of these natural building blocks. These amino acids have unique chemical or physical properties as a result of their specific side chain residues. Their incorporation into proteins through ribosomal translation in response to one of the stop codons has opened a new way to manipulate and study proteins by enabling new functionalities, thus expending the genetic code. Different unnatural amino acids have different functionalities, hence, the ability to incorporate two different unnatural amino acids, in response to two different stop codons into one protein is a useful tool in protein manipulation. This ability has been achieved previously only in in vivo translational systems, however, with limited functionality. Herein, we report the incorporation of two different unnatural amino acids in response to two different stop codons into one protein, utilizing a cell-free protein synthesis system. Biotechnol. Bioeng. 2017;114: 1065-1073. © 2016 Wiley Periodicals, Inc.
authors.affiliation: Department of Life Sciences and Ilse Katz Institute for Nanoscale Science and Technology, Ben-Gurion University of the Negev, P. O. Box 653, 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, P. O. Box 653, 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, P. O. Box 653, Beer-Sheva, 84105, Israel.
authors.forename: Orr
authors.initials: O
authors.lastname: Schlesinger
authors.affiliation: Department of Chemical Physics, Weizmann Institute of Science, Rehovot, Israel.
authors.forename: Haim Yuval
authors.initials: HY
authors.lastname: Aviram
authors.affiliation: Department of Chemical Physics, Weizmann Institute of Science, Rehovot, Israel.
authors.forename: Inbal
authors.initials: I
authors.lastname: Riven
authors.affiliation: Department of Chemical Physics, Weizmann Institute of Science, Rehovot, Israel.
authors.forename: Gilad
authors.initials: G
authors.lastname: Haran
authors.affiliation: Department of Life Sciences and Ilse Katz Institute for Nanoscale Science and Technology, Ben-Gurion University of the Negev, P. O. Box 653, Beer-Sheva, 84105, Israel.
authors.forename: Lital
authors.initials: L
authors.lastname: Alfonta
mesh_terms: D000596:Amino Acids / Q000737:chemistry / Q000378:metabolism*; D002457:Cell Extracts; D002474:Cell-Free System / Q000378:metabolism*; D018388:Codon, Terminator / Q000378:metabolism*; D004926:Escherichia coli; D031541:Fluorescence Resonance Energy Transfer; D014176:Protein Biosynthesis; D015202:Protein Engineering / Q000379:methods*
chemical_list: D000596:Amino Acids; D002457:Cell Extracts; D018388:Codon, Terminator
keywords: Förster resonance energy transfer; cell-free protein synthesis; dual genetic code expansion; orthogonal translation systems