title: Optimisation of a system for the co-translational incorporation of a keto amino acid and its application to a tumour-specific Anticalin.
abstract: The bioorthogonal keto group has attracted interest for the site-specific chemical conjugation of recombinant proteins under mild conditions, e.g. with aminooxy-functionalised fluorescent probes, radiometal chelates, toxins or polymers. However, the cotranslational incorporation of the corresponding non-canonical amino acid p-acetyl-L-phenylalanine (Apa) into proteins expressed in Escherichia coli by means of amber suppression using a previously described system with a mutated tRNA and an engineered tyrosyl-tRNA synthetase from Methanococcus jannaschii shows limited efficiency and considerable promiscuity towards endogenous amino acids. Employing a one-plasmid system that encodes all three components required for selection, i.e. the modified aminoacyl-tRNA synthetase (aaRS), the cognate amber suppressor tRNA and the enhanced green fluorescent protein equipped with an amber stop codon and serving as reporter, we have generated an Apa-specific aaRS&tRNA pair with considerably improved efficiency (17-fold increased expression) and also fidelity (6-fold). To this end, both the aaRS and the tRNA were subjected to doped random mutagenesis and selection in altogether four evolutionary cycles using fluorescence-activated bacterial cell sorting as well as automated screening of microcultures. The resulting aaRS&tRNA pair was applied to the functionalisation of an Anticalin with specificity towards oncofetal fibronectin by introducing a keto group at a permissible site for subsequent conjugation with a fluorescent dye, thus allowing visualisation of this tumour target under the microscope.
authors.affiliation: Munich Center for Integrated Protein Science (CIPS-M) and Lehrstuhl für Biologische Chemie, Technische Universität München, 85350 Freising-Weihenstephan, Germany.
authors.forename: Andreas J
authors.initials: AJ
authors.lastname: Reichert
authors.affiliation: Munich Center for Integrated Protein Science (CIPS-M) and Lehrstuhl für Biologische Chemie, Technische Universität München, 85350 Freising-Weihenstephan, Germany.
authors.forename: Gabriele
authors.initials: G
authors.lastname: Poxleitner
authors.affiliation: Munich Center for Integrated Protein Science (CIPS-M) and Lehrstuhl für Biologische Chemie, Technische Universität München, 85350 Freising-Weihenstephan, Germany.
authors.forename: Martin
authors.initials: M
authors.lastname: Dauner
authors.affiliation: Munich Center for Integrated Protein Science (CIPS-M) and Lehrstuhl für Biologische Chemie, Technische Universität München, 85350 Freising-Weihenstephan, Germany skerra@tum.de.
authors.forename: Arne
authors.initials: A
authors.lastname: Skerra
mesh_terms: D000970:Antineoplastic Agents / Q000737:chemistry / Q000378:metabolism*; D004926:Escherichia coli / Q000235:genetics; D010649:Phenylalanine / Q000031:analogs & derivatives* / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D015202:Protein Engineering / Q000379:methods*; D046188:Protein Modification, Translational / Q000235:genetics*; D012343:RNA, Transfer / Q000235:genetics / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*
chemical_list: C476995:3-(4-acetylphenyl)-2-aminopropanoic acid; D000970:Antineoplastic Agents; D011994:Recombinant Proteins; D010649:Phenylalanine; D012343:RNA, Transfer
keywords: genetic code expansion; keto-functionalisation; non-canonical amino acid; synthetic biology; unnatural amino acid