Tags

Type your tag names separated by a space and hit enter

Optimisation of a system for the co-translational incorporation of a keto amino acid and its application to a tumour-specific Anticalin.
Protein Eng Des Sel. 2015 Dec; 28(12):553-65.PE

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+Show Affiliations

Munich Center for Integrated Protein Science (CIPS-M) and Lehrstuhl für Biologische Chemie, Technische Universität München, 85350 Freising-Weihenstephan, Germany.Munich Center for Integrated Protein Science (CIPS-M) and Lehrstuhl für Biologische Chemie, Technische Universität München, 85350 Freising-Weihenstephan, Germany.Munich Center for Integrated Protein Science (CIPS-M) and Lehrstuhl für Biologische Chemie, Technische Universität München, 85350 Freising-Weihenstephan, Germany.Munich Center for Integrated Protein Science (CIPS-M) and Lehrstuhl für Biologische Chemie, Technische Universität München, 85350 Freising-Weihenstephan, Germany skerra@tum.de.

Pub Type(s)

Journal Article
Research Support, Non-U.S. Gov't

Language

eng

PubMed ID

26405058

Citation

Reichert, Andreas J., et al. "Optimisation of a System for the Co-translational Incorporation of a Keto Amino Acid and Its Application to a Tumour-specific Anticalin." Protein Engineering, Design & Selection : PEDS, vol. 28, no. 12, 2015, pp. 553-65.
Reichert AJ, Poxleitner G, Dauner M, et al. Optimisation of a system for the co-translational incorporation of a keto amino acid and its application to a tumour-specific Anticalin. Protein Eng Des Sel. 2015;28(12):553-65.
Reichert, A. J., Poxleitner, G., Dauner, M., & Skerra, A. (2015). Optimisation of a system for the co-translational incorporation of a keto amino acid and its application to a tumour-specific Anticalin. Protein Engineering, Design & Selection : PEDS, 28(12), 553-65. https://doi.org/10.1093/protein/gzv048
Reichert AJ, et al. Optimisation of a System for the Co-translational Incorporation of a Keto Amino Acid and Its Application to a Tumour-specific Anticalin. Protein Eng Des Sel. 2015;28(12):553-65. PubMed PMID: 26405058.
* Article titles in AMA citation format should be in sentence-case
TY - JOUR T1 - Optimisation of a system for the co-translational incorporation of a keto amino acid and its application to a tumour-specific Anticalin. AU - Reichert,Andreas J, AU - Poxleitner,Gabriele, AU - Dauner,Martin, AU - Skerra,Arne, Y1 - 2015/09/23/ PY - 2015/08/16/received PY - 2015/08/17/accepted PY - 2015/9/26/entrez PY - 2015/9/26/pubmed PY - 2016/5/26/medline KW - genetic code expansion KW - keto-functionalisation KW - non-canonical amino acid KW - synthetic biology KW - unnatural amino acid SP - 553 EP - 65 JF - Protein engineering, design & selection : PEDS JO - Protein Eng. Des. Sel. VL - 28 IS - 12 N2 - 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. SN - 1741-0134 UR - https://www.unboundmedicine.com/medline/citation/26405058/Optimisation_of_a_system_for_the_co_translational_incorporation_of_a_keto_amino_acid_and_its_application_to_a_tumour_specific_Anticalin_ L2 - https://academic.oup.com/peds/article-lookup/doi/10.1093/protein/gzv048 DB - PRIME DP - Unbound Medicine ER -