title: Cell-free expression with the toxic amino acid canavanine.
abstract: Canavanine is a naturally occurring noncanonical amino acid, which is analogous to arginine. It is a potent antimetabolite and natural allelochemic agent, capable of affecting or blocking regulatory and catalytic reactions that involve arginine. Incorporated into proteins at arginine positions, canavanine can be detrimental to protein stability and functional integrity. Although incorporation of canavanine into proteins has long been documented, due to its toxicity, expression in Escherichia coli and other common hosts remains a considerable challenge. Here, we present a simple, cell-free expression system with markedly improved performance compared to heterologous expression. The cell-free expression system does not require any tuning besides substitution of arginine by canavanine. We show that our technique enables highly efficient protein expression in small volumes with arginine being fully replaced by canavanine for functional and structural studies.
authors.affiliation: Universität des Saarlandes, Department of Experimental Physics, Campus Saarbruecken, 66123 Saarbruecken, Germany.
authors.forename: Emanuel G
authors.initials: EG
authors.lastname: Worst
authors.affiliation: Technische Universität Berlin, Institute of Chemistry, Mueller-Breslau-Strasse 10, 10623 Berlin, Germany.
authors.forename: Matthias P
authors.initials: MP
authors.lastname: Exner
authors.affiliation: Technische Universität Berlin, Institute of Chemistry, Mueller-Breslau-Strasse 10, 10623 Berlin, Germany.
authors.forename: Alessandro
authors.initials: A
authors.lastname: De Simone
authors.affiliation: Universität des Saarlandes, Department of Experimental Physics, Campus Saarbruecken, 66123 Saarbruecken, Germany.
authors.forename: Marc
authors.initials: M
authors.lastname: Schenkelberger
authors.affiliation: University of Minnesota, School of Physics and Astronomy, Minneapolis, MN 55455, USA.
authors.forename: Vincent
authors.initials: V
authors.lastname: Noireaux
authors.affiliation: Technische Universität Berlin, Institute of Chemistry, Mueller-Breslau-Strasse 10, 10623 Berlin, Germany.
authors.forename: Nediljko
authors.initials: N
authors.lastname: Budisa
authors.affiliation: Universität des Saarlandes, Department of Experimental Physics, Campus Saarbruecken, 66123 Saarbruecken, Germany. Electronic address: albrecht.ott@physik.uni-saarland.de.
authors.forename: Albrecht
authors.initials: A
authors.lastname: Ott
mesh_terms: D019943:Amino Acid Substitution; D001120:Arginine / Q000737:chemistry / Q000378:metabolism; D002172:Canavanine / Q000737:chemistry / Q000378:metabolism*; D002474:Cell-Free System; D004926:Escherichia coli / Q000235:genetics; D049452:Green Fluorescent Proteins / Q000737:chemistry / Q000378:metabolism; D015202:Protein Engineering / Q000379:methods*; D011994:Recombinant Proteins / Q000737:chemistry* / Q000378:metabolism*
chemical_list: D011994:Recombinant Proteins; C485184:enhanced green fluorescent protein; D049452:Green Fluorescent Proteins; D002172:Canavanine; D001120:Arginine
keywords: Antimetabolite; Arginine; Canavanine; Cell-free expression; Noncanonical amino acid