Synthesis and site-directed fluorescence labeling of azido proteins using eukaryotic cell-free orthogonal translation systems.
Eukaryotic cell-free systems based on wheat germ and Spodoptera frugiperda insect cells were equipped with an orthogonal amber suppressor tRNA-synthetase pair to synthesize proteins with a site-specifically incorporated p-azido-l-phenylalanine residue in order to provide their chemoselective fluorescence labeling with azide-reactive dyes by Staudinger ligation. The specificity of incorporation and bioorthogonality of labeling within complex reaction mixtures was shown by means of translation and fluorescence detection of two model proteins: β-glucuronidase and erythropoietin. The latter contained the azido amino acid in proximity to a signal peptide for membrane translocation into endogenous microsomal vesicles of the insect cell-based system. The results indicate a stoichiometric incorporation of the azido amino acid at the desired position within the proteins. Moreover, the compatibility of cotranslational protein translocation, including glycosylation and amber suppression-based incorporation of p-azido-l-phenylalanine within a cell-free system, is demonstrated. The presented approach should be particularly useful for providing eukaryotic and membrane-associated proteins for investigation by fluorescence-based techniques.
Fraunhofer Institute for Biomedical Engineering (IBMT), Branch Potsdam-Golm, 14476 Potsdam, Germany.
Robert B
RB
Quast
RiNA, 12489 Berlin, Germany.
Iris
I
Claussnitzer
RiNA, 12489 Berlin, Germany.
Helmut
H
Merk
Fraunhofer Institute for Biomedical Engineering (IBMT), Branch Potsdam-Golm, 14476 Potsdam, Germany.
Stefan
S
Kubick
RiNA, 12489 Berlin, Germany. Electronic address: gerrits@rina-gmbh.eu.
Michael
M
Gerrits
D000604:Amino Acyl-tRNA Synthetases / Q000378:metabolism; D000818:Animals; D001386:Azides / Q000737:chemistry*; D002474:Cell-Free System; D004591:Electrophoresis, Polyacrylamide Gel; D004921:Erythropoietin / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D005456:Fluorescent Dyes / Q000737:chemistry*; D005966:Glucuronidase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D010649:Phenylalanine / Q000737:chemistry / Q000378:metabolism; D061987:Sf9 Cells / Q000378:metabolism; D014908:Triticum / Q000378:metabolism
D001386:Azides; D005456:Fluorescent Dyes; D004921:Erythropoietin; D010649:Phenylalanine; D005966:Glucuronidase; D000604:Amino Acyl-tRNA Synthetases
Cell-free protein synthesis; Eukaryotic; Microsomal vesicles; Site-directed fluorescence labeling; Staudinger ligation; Translocation