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Engineering the Genetic Code of Escherichia Coli with Methionine Analogues and Bioorthogonal Amino Acids for Protein Immobilization

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Engineering the Genetic Code of Escherichia Coli with Methionine Analogues and Bioorthogonal Amino Acids for Protein Immobilization Engineering the genetic code of Escherichia coli with methionine analogues and bioorthogonal amino acids for protein immobilization vorgelegt von M. Sc. in Biotechnologie Alessandro De Simone aus Bari, Italien Von der Fakultät II – Mathematik und Naturwissenschaften der Technischen Universität Berlin zur Erlangung des akademischen Grades Doktor der Naturwissenschaften Dr. rer. nat. genehmigte Dissertation Promotionsausschuss: Vorsitzender: Prof. Dr. Thomas Friedrich Erstgutachter: Prof. Dr. Nediljko Budisa Zweitgutachter: Prof. Dr. Christian Hackenberger Tag der wissenschaftlichen Aussprache: 17.06.2016 Berlin 2016 to my family “Mystics understand the roots of the Tao but not its branches; scientists understand its branches but not its roots. Science does not need mysticism and mysticism does not need science; but man needs both.” – Fritjof Capra Parts of this work have been submitted to publication: De Simone A, Hoesl MG, Acevedo-Rocha CG, Budisa N. Towards reassignment of the AUG Methionine codon to two different non-canonical amino acids in bacterial translation. 2016 Croatica Chemica Acta (under revision). De Simone A, Hoesl MG, Budisa N. Engineering Lipases with an expanded genetic code. 2016 Applied Biocatalysis: From Fundamental Science to Industrial Applications Wiley- VCH (ISBN 978-3-527-33669-2) (in press). Other scientific publications: Worst EG, Exner MP, De Simone A, Schenkelberger M, Noireaux V, Budisa N, Ott A. Cell- free expression with the toxic amino acid canavanine. 2015 Bioorg. Med. Chem. Lett. 25, 3658–60 Poster presentations: De Simone A, Budisa N. Incorporation of bioorthogonal handles into proteins. 2014 3rd International Symposium of the Collaborative Research Center (SFB) 765 on “Multivalency and Chemistry and Biochemistry”, Freie Universität Berlin, Germany. I Table of contents Table of contents Table of contents ................................................................................................................... I List of figures ..................................................................................................................... IV List of tables ...................................................................................................................... VII Summary ...............................................................................................................................1 Zusammenfassung ................................................................................................................2 Abbreviations and definitions ..............................................................................................3 1. Simultaneous reassigment of the AUG codon to two Met analogues ..............................8 1.1 Introduction................................................................................................................8 1.1.1 The genetic code: origin and evolution ...............................................................8 1.1.2 An overview of protein translation ................................................................... 12 1.1.3 Expanding the genetic code beyond the canonical 20 amino acids .................... 15 1.1.4 Residue-specific incorporation of non-canonical amino acids ........................... 16 1.1.5 Site-specific incorporation of non-canonical amino acids ................................. 17 1.1.6 Sense codon reassignment: breaking codon degeneracy ................................... 22 1.1.7 Reassignment of the AUG codon to methionine analogues ............................... 23 1.1.8 Combination of site-specific and residue-specific methods for AUG codon reassignment ................................................................................................................. 26 1.2 Aim of the study ....................................................................................................... 28 1.3 Results and discussion .............................................................................................. 29 Met 1.3.1 Search for an orthogonal MetRS/tRNA pair ................................................. 29 Met 1.3.2 The SaMetRS/SatRNA pair.......................................................................... 30 Met 1.3.3 Assembly of pMEc vector with MetRS/tRNA pairs...................................... 35 1.3.4 AUG reassignment by SaMetRS overexpression .............................................. 37 Met 1.3.5 Knockout of elongator tRNA in E. coli MG1655.......................................... 41 Met 1.3.6 Knockout of elongator tRNA in E. coli B834(DE3) ..................................... 44 Met 1.3.7 AUG reassignment in absence of elongator tRNA ........................................ 45 Pyl 1.3.8 A different strategy for AUG decoding: the PylRS/tRNA pair ...................... 48 1.3.9 Other rescue strategies for AUG reassignment ................................................. 56 1.4 Conclusions and Outlook ......................................................................................... 61 2. Site-specific bioconjugation and immobilization of lipase ............................................ 64 2.1 Introduction.............................................................................................................. 64 2.1.1 Lipase: an important industrial biocatalyst ....................................................... 64 2.1.2 Lipase improvement by global incorporation of ncAAs .................................... 67 2.1.3 Lipase improvement by site-specific modifications .......................................... 69 2.1.4 Lipase improvement by immobilization ........................................................... 70 2.1.5 “Click” chemistry for enzyme immobilization .................................................. 71 2.2 Aim of the study ....................................................................................................... 73 2.3 Results and discussion .............................................................................................. 74 2.3.1 Screening of permissive sites on protein surface ............................................... 74 2.3.2 Comparison of suppression efficiency at position K25TAG and E108TAG ...... 75 Table of contents II 2.3.3 Comparison of suppression efficiency between BL21 and JX33 strains ............ 76 2.3.4 Suppression of E108TAG with Plk ................................................................... 76 2.3.5 Expression, purification and evaluation of the activity of TTL(E108Plk) .......... 78 2.3.6 Conjugation of TTL(M83L/E108Plk) with azide-biotin via CuAAC ................ 79 ® 2.3.7 Immobilization of biotinyl-TTL on Strep-Tactin beads ................................... 81 2.3.8 Immobilization of alkynyl-TTL on azide-agarose beads ................................... 82 2.3.9 Conjugation of TTL(M83L/K25Plk) and TTL(M83L/D221Plk) ....................... 84 2.3.10 Suppression of single and multiple amber codons with CooK ......................... 86 2.3.11 Incorporation of CooK into TTL at position D221TAG .................................. 87 2.3.12 Incorporation of CooK into TTL at position E108TAG .................................. 88 2.4 Conclusions and Outlook ......................................................................................... 90 3. Materials ......................................................................................................................... 93 3.1 Technical equipment .................................................................................................. 93 3.2 Chemicals .................................................................................................................. 95 3.3 Media and supplements .............................................................................................. 95 3.4 Strains ........................................................................................................................ 99 3.5 Primers .................................................................................................................... 101 3.6 Synthetic DNA constructs ........................................................................................ 102 3.7 PCR products for pMEc assembly ............................................................................ 102 3.8 Plasmids................................................................................................................... 104 3.9 Enzymes .................................................................................................................. 105 3.10 Kits ........................................................................................................................ 107 3.11 Buffers and solutions .............................................................................................. 107 4. Methods ......................................................................................................................... 111 4.1 Molecular biological methods .................................................................................. 111 4.1.1 Isolation of plasmid DNA .............................................................................. 111 4.1.2 Polymerase chain reaction .............................................................................. 111 4.1.3 Cloning .......................................................................................................... 113 4.2 Microbiological methods .........................................................................................
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