Cell-free synthesis of functional human epidermal growth factor receptor: Investigation of ligand-independent dimerization in Sf21 microsomal membranes using non-canonical amino acids.
Cell-free protein synthesis systems represent versatile tools for the synthesis and modification of human membrane proteins. In particular, eukaryotic cell-free systems provide a promising platform for their structural and functional characterization. Here, we present the cell-free synthesis of functional human epidermal growth factor receptor and its vIII deletion mutant in a microsome-containing system derived from cultured Sf21 cells. We provide evidence for embedment of cell-free synthesized receptors into microsomal membranes and asparagine-linked glycosylation. Using the cricket paralysis virus internal ribosome entry site and a repetitive synthesis approach enrichment of receptors inside the microsomal fractions was facilitated thereby providing analytical amounts of functional protein. Receptor tyrosine kinase activation was demonstrated by monitoring receptor phosphorylation. Furthermore, an orthogonal cell-free translation system that provides the site-directed incorporation of p-azido-L-phenylalanine is characterized and applied to investigate receptor dimerization in the absence of a ligand by photo-affinity cross-linking. Finally, incorporated azides are used to generate stable covalently linked receptor dimers by strain-promoted cycloaddition using a novel linker system.
Fraunhofer Institute for Cell Therapy and Immunology (IZI), Branch Bioanalytics and Bioprocesses (IZI-BB), Am Mühlenberg 13, D-14476 Potsdam, Germany.
Robert B
RB
Quast
Institute for Macromolecular Chemistry, University of Freiburg, Stefan-Meier-Str. 31, D-79104 Freiburg, Germany.|Freiburg Institute for Advanced Studies (FRIAS), School of Soft Matter Research, University of Freiburg, Albertstr. 19, D-79104 Freiburg, Germany.|Center for Biosystems Analysis (ZBSA), University of Freiburg, Habsburger Str. 49, D-79104 Freiburg, Germany.
Biljana
B
Ballion
Fraunhofer Institute for Cell Therapy and Immunology (IZI), Branch Bioanalytics and Bioprocesses (IZI-BB), Am Mühlenberg 13, D-14476 Potsdam, Germany.
Marlitt
M
Stech
Fraunhofer Institute for Cell Therapy and Immunology (IZI), Branch Bioanalytics and Bioprocesses (IZI-BB), Am Mühlenberg 13, D-14476 Potsdam, Germany.
Andrei
A
Sonnabend
Chemical Biology Research Group, Hungarian Academy of Sciences, CNS, IOC, Magyar tudósok krt. 2, H-1117 Budapest, Hungary.
Balázs R
BR
Varga
Fraunhofer Institute for Cell Therapy and Immunology (IZI), Branch Bioanalytics and Bioprocesses (IZI-BB), Am Mühlenberg 13, D-14476 Potsdam, Germany.
Doreen A
DA
Wüstenhagen
Chemical Biology Research Group, Hungarian Academy of Sciences, CNS, IOC, Magyar tudósok krt. 2, H-1117 Budapest, Hungary.
Péter
P
Kele
Institute for Macromolecular Chemistry, University of Freiburg, Stefan-Meier-Str. 31, D-79104 Freiburg, Germany.|Freiburg Institute for Advanced Studies (FRIAS), School of Soft Matter Research, University of Freiburg, Albertstr. 19, D-79104 Freiburg, Germany.|Center for Biosystems Analysis (ZBSA), University of Freiburg, Habsburger Str. 49, D-79104 Freiburg, Germany.
Stefan M
SM
Schiller
Fraunhofer Institute for Cell Therapy and Immunology (IZI), Branch Bioanalytics and Bioprocesses (IZI-BB), Am Mühlenberg 13, D-14476 Potsdam, Germany.
Stefan
S
Kubick
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