title: Probing unnatural amino acid integration into enhanced green fluorescent protein by genetic code expansion with a high-throughput screening platform.
abstract: 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). 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/tRNAPylCUApair originating fromMethanosarcina barkeriinE. coli. The site-specific uAA functionalization was confirmed by LC-MS/MS analysis. uAA-eGFP production and biomass growth in parallelizedE. colicultivations 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.
  
  
  CONCLUSIONS
  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/tRNAPylCUApairs as well as further optimization of culture conditions.
authors.affiliation: AVT, Biochemical Engineering, RWTH Aachen University, Aachen, 52074 Germany.
authors.forename: Georg
authors.initials: G
authors.lastname: Wandrey
authors.affiliation: Institute for Pharmacy and Food Chemistry, University of Würzburg, Am Hubland, Würzburg, 97074 Germany.
authors.forename: Joel
authors.initials: J
authors.lastname: Wurzel
authors.affiliation: AVT, Biochemical Engineering, RWTH Aachen University, Aachen, 52074 Germany.
authors.forename: Kyra
authors.initials: K
authors.lastname: Hoffmann
authors.affiliation: AVT, Biochemical Engineering, RWTH Aachen University, Aachen, 52074 Germany.
authors.forename: Tobias
authors.initials: T
authors.lastname: Ladner
authors.affiliation: AVT, Biochemical Engineering, RWTH Aachen University, Aachen, 52074 Germany.
authors.forename: Jochen
authors.initials: J
authors.lastname: Büchs
authors.affiliation: Institute for Pharmacy and Food Chemistry, University of Würzburg, Am Hubland, Würzburg, 97074 Germany.
authors.forename: Lorenz
authors.initials: L
authors.lastname: Meinel
authors.affiliation: Institute for Pharmacy and Food Chemistry, University of Würzburg, Am Hubland, Würzburg, 97074 Germany.
authors.forename: Tessa
authors.initials: T
authors.lastname: Lühmann
keywords: Amber codon suppression; Bio-orthogonal chemistry; High-throughput screening; Online monitoring system; Protein engineering; Unnatural amino acid