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Expanding the Toolkit of Protein Engineering: Towards Multiple Simultaneous in Vivo Incorporation of Noncanonical Amino Acids

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Expanding the Toolkit of Protein Engineering: Towards Multiple Simultaneous in Vivo Incorporation of Noncanonical Amino Acids TECHNISCHE UNIVERSITÄT MÜNCHEN Max-Planck-Institut für Biochemie Expanding the Toolkit of Protein Engineering: Towards Multiple Simultaneous In Vivo Incorporation of Noncanonical Amino Acids Michael G. Hösl Vollständiger Abdruck der von der Fakultät für Chemie der Technischen Universität München zur Erlangung des akademischen Grades eines Doktors der Naturwissenschaften genehmigten Dissertation. Vorsitzender: Univ.-Prof. Dr. Michael Groll Prüfer der Dissertation: 1. Univ.-Prof. Dr. Nediljko Budisa, Technische Universität Berlin 2. Univ.-Prof. Dr. Thomas Kiefhaber 3. Univ.-Prof. Dr. Johannes Buchner Die Dissertation wurde am 01.02.11 bei der Technischen Universität München eingereicht und durch die Fakultät für Chemie am 03.03.11 angenommen. To Teresa Ariadna García-Grajalva Lucas who influenced the idea of TAG → AGG switch just by the existence of her name Sleeping is giving in, no matter what the time is. Sleeping is giving in, so lift those heavy eyelids. People say that you'll die faster than without water. But we know it's just a lie, scare your son, scare your daughter. People say that your dreams are the only things that save ya. Come on baby in our dreams, we can live on misbehavior. The Arcade Fire Parts of this work were published as listed below: Hoesl, MG, Budisa, N. Expanding and engineering the genetic code in a single expression experiment. ChemBioChem 2011, 12, 552-555. Further publications: Hoesl, MG*, Staudt, H*, Dreuw, A, Budisa, N, Grininger, M, Oesterhelt, D, Wachtveitl, J. Manipulating the eletron transfer in Dodecin by isostructual noncanonical Trp analogs. 2011, [in preparation]. *authors contributed equally to this work Nehring, S*, Hoesl, MG*, Acevedo-Rocha, CG*, Royter, M, Wolschner, C, Wiltschi, B, Budisa, N, Antranikian, G. Effects of additives on the activity of TTL congeners. 2011, [in preparation]. *authors contributed equally to this work Hoesl, MG*, Acevedo-Rocha, CG*, Nehring, S*, Royter, M, Wolschner, C, Wiltschi, B, Budisa, N, Antranikian, G. Lipase Congeners Designed by Genetic Code Engineering. ChemCatChem 2011, 3, 213-221. *authors contributed equally to this work Hoesl, MG, Larregola, M, Cui, H, Budisa, N. Azatryptophans as tools to study polarity requirements for folding of green fluorescent protein. J. Pept. Sci. 2010, 16, 589-595. Merkel, L, Hoesl, MG, Albrecht, M, Schmidt, A, Budisa, N. Blue Fluorescent Amino Acids As In Vivo Building Blocks for Proteins. ChemBioChem 2010, 11, 305-314. Lepthien, S, Hoesl, MG, Merkel, L, Budisa, N. Azatryptophans endow proteins with intrinsic blue fluorescence. Proc. Natl. Acad. Sci. U.S.A. 2008, 105, 16095-16100. Review articles: Hoesl, MG, Merkel, L, Budisa, N. Synthetic biology of autofluorescent proteins, in Fluorescent Proteins – from Fundamental Research to Bioanalytics (Ed.: G. Jung), Springer Verlag, Heidelberg, 2011, p. [in press]. Hoesl, MG, Budisa, N. In vivo incorporation of multiple non-canonical amino acids into proteins. Angew. Chem. Int. Ed. Engl. 2011, [epub, DOI: 10.1002/anie.201005680]. Hoesl, MG, Budisa, N. Nicht-kanonische Aminosäuren in der Synthetischen Biologie. BIOspektrum 2010, 16. Jahrgang, 309-311. Poster presentations: Hoesl, MG, Lepthien, S, Merkel, L, Budisa, N, (2009). Azatryptophans endow proteins with intrinsic blue fluorescence. 3rd European Conference on Chemistry for Life Sciences, Sep 2 - 5, 2009, Frankfurt/Main, Germany. Oral presentations: Kongenere Lipasen mit verbesserter katalytischer Aktivität und Substratzugang. 3. Annual meeting of the Biokatalyse2021 Cluster, Oct 18 – 19, 2010, Kiel, Germany. Azatryptophans endow Proteins with Intrinsic Blue Fluorescence (oral poster presentation). 3rd European Conference on Chemistry for Life Sciences, Sep 2 – 5, 2009, Frankfurt/Main, Germany. Department Retreat, Trp Codon Reassignment in the Flavoprotein Dodecin, Nov 09 – 12, 2008, Ringberg Castle, Ringberg, Germany. 8. Graduate Retreat, Design of Blue Fluorescent Proteins with an Expanded Genetic Code, Jun 28 - 30, 2008, Ringberg Castle, Ringberg, Germany. 7. Graduate Retreat, In vivo and in vitro incorporation of tryptophan analogs into ECFP and Annexin V, Jun 15 – 18, 2007, Ringberg Castle, Ringberg, Germany. Table of contents I I Table of contents I Table of contents ....................................................................................................... I II Summary .................................................................................................................. V III Zusammenfassung ................................................................................................... VI IV Abbreviations and definitions ................................................................................. VII IV.I Definitions ....................................................................................................................... VII IV.II Abbreviations ................................................................................................................. VIII 1 Introduction ............................................................................................................. 1 1.1 The Genetic Code .............................................................................................................. 1 1.2 Protein translation ............................................................................................................ 4 1.2.1 General aspects of protein translation .......................................................................... 4 1.2.2 Aminoacyl-tRNA synthetases ........................................................................................ 6 1.2.3 Transfer RNAs ................................................................................................................ 9 1.2.4 Translation termination versus termination suppression ........................................... 11 1.3 Synthetic evolution of the genetic code ......................................................................... 13 1.3.1 In vitro semi-synthetic incorporation .......................................................................... 13 1.3.2 Genetic code engineering ........................................................................................... 14 1.3.3 Genetic code expansion .............................................................................................. 18 1.3.4 Comparison of synthetic evolution methods of the genetic code .............................. 23 1.4 Aim of this study: Expanding the toolkit of protein engineering .................................... 25 2 Results and Discussion ............................................................................................ 26 2.1 The aminoacyl-tRNA synthetase:suppressor tRNA pairs ................................................ 26 Tyr 2.1.1 mjBpaRS:mjtRNA CUA ................................................................................................. 26 Phe 2.1.2 scPheRS(T415G):sctRNA CUA_UG ................................................................................ 27 2.2 Characterization of mjBpaRS and scPheRS(T415G) ........................................................ 28 2.2.1 In vitro analysis of noncanonical amino acid activation .............................................. 29 2.2.2 In vivo analysis of noncanonical amino acid incorporation ........................................ 30 2.2.3 Evaluation of amber suppression rates ....................................................................... 39 2.3 A new vector system for aaRS:tRNA coexpression ......................................................... 44 2.3.1 Design strategy for the pMEc vector family ................................................................ 44 2.3.2 Assembly of pMEc vectors with aaRS:tRNACUA pairs ................................................... 45 2.3.3 Evaluation of assembled pMEc vectors ....................................................................... 47 II Table of contents 2.4 Simultaneous genetic code expansion and engineering ................................................. 50 2.4.1 Simultaneous incorporation of Hpg and Bpa into -b* .............................................. 50 2.4.2 Simultaneous incorporation of (4S-F)Pro and Bpa into EGFP ...................................... 51 2.4.3 Simultaneous incorporation of Nle or Aha and Bpa into TTL ...................................... 55 2.4.4 Evaluation of protein yield in combined SPI and SCS experiments ............................. 62 2.4.5 Dependence of suppression rate on mRNA sequence context ................................... 63 2.4.6 Does SPI promote amber suppression? ....................................................................... 65 2.4.7 Summary and concluding remarks .............................................................................. 66 2.5 Towards AGG codon reassignment for ncaa incorporation ............................................ 67 3 Conclusions and outlook ......................................................................................... 71 3.1 Emancipation of the amber stop codon .......................................................................... 71 3.2 Emancipation of the AGG codon ..................................................................................... 72 4 Materials ................................................................................................................ 75 4.1 Chemicals ........................................................................................................................
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