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Probing unnatural amino acid integration into enhanced green fluorescent protein by genetic code expansion with a high-throughput screening platform

Please always quote using this URN: urn:nbn:de:bvb:20-opus-166304
  • Background Genetic code expansion has developed into an elegant tool to incorporate unnatural amino acids (uAA) at predefined sites in the protein backbone in response to an amber codon. However, recombinant production and yield of uAA comprising proteins are challenged due to the additional translation machinery required for uAA incorporation. Results We developed a microtiter plate-based high-throughput monitoring system (HTMS) to study and optimize uAA integration in the model protein enhanced green fluorescence protein (eGFP). TwoBackground Genetic code expansion has developed into an elegant tool to incorporate unnatural amino acids (uAA) at predefined sites in the protein backbone in response to an amber codon. However, recombinant production and yield of uAA comprising proteins are challenged due to the additional translation machinery required for uAA incorporation. Results We developed a microtiter plate-based high-throughput monitoring system (HTMS) to study and optimize uAA integration in the model protein enhanced green fluorescence protein (eGFP). Two uAA, propargyl-L-lysine (Plk) and (S)-2-amino-6-((2-azidoethoxy) carbonylamino) hexanoic acid (Alk), were incorporated at the same site into eGFP co-expressing the native PylRS/tRNAPyl CUA pair originating from Methanosarcina barkeri in E. coli. The site-specific uAA functionalization was confirmed by LC-MS/MS analysis. uAA-eGFP production and biomass growth in parallelized E. coli cultivations was correlated to (i) uAA concentration and the (ii) time of uAA addition to the expression medium as well as to induction parameters including the (iii) time and (iv) amount of IPTG supplementation. The online measurements of the HTMS were consolidated by end point-detection using standard enzyme-linked immunosorbent procedures. Conclusion The developed HTMS is powerful tool for parallelized and rapid screening. In light of uAA integration, future applications may include parallelized screening of different PylRS/tRNAPyl CUA pairs as well as further optimization of culture conditions.show moreshow less

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Metadaten
Author: Georg Wandrey, Joel Wurzel, Kyra Hoffmann, Tobias Ladner, Jochen Büchs, Lorenz Meinel, Tessa Lühmann
URN:urn:nbn:de:bvb:20-opus-166304
Document Type:Journal article
Faculties:Fakultät für Chemie und Pharmazie / Institut für Pharmazie und Lebensmittelchemie
Language:English
Parent Title (English):Journal of Biological Engineering
Year of Completion:2016
Volume:10
Issue:11
Source:Journal of Biological Engineering (2016) 10:11 DOI 10.1186/s13036-016-0031-6
DOI:https://doi.org/10.1186/s13036-016-0031-6
Dewey Decimal Classification:6 Technik, Medizin, angewandte Wissenschaften / 61 Medizin und Gesundheit / 610 Medizin und Gesundheit
Tag:amber codon suppression; bio-orthogonal chemistry; high-throughput screening; online monitoring system; protein engineering; unnatural amino acid
Release Date:2018/11/14
EU-Project number / Contract (GA) number:296679
OpenAIRE:OpenAIRE
Licence (German):License LogoCC BY: Creative-Commons-Lizenz: Namensnennung 4.0 International