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Cell-free synthesis of functional human epidermal growth factor receptor: Investigation of ligand-independent dimerization in Sf 21 microsomal membranes using non-canonical amino acids

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ABSTRACT

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.

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Enrichment of functional EGFR-eYFP in the Sf21 microsomal fraction by repetitive cell-free synthesis.(a) Total protein yields after each cell-free reaction in the complete reaction mixture (R), the supernatant fraction (S) and the microsomal fraction (M). Error bars represent the standard deviation of triplicate analysis. (b) Autoradiography of corresponding samples after electrophoretic separation under denaturing conditions. (c) Yields of total protein and eYFP fluorescence after each cell-free reaction relative to the first reaction. (d) Confocal fluorescence image of EGFR-eYFP in microsomal fraction taken under hypoosmotic buffer conditions. (e) Western Blot of microsomal fractions with and without EGFR-eYFP after incubation in kinase buffer. Isotopic labeling was achieved by 14C-leucine supplementation. The western blots (e) have been adapted in contrast, brightness and sharpness for better visibility. The original images can be found in Supplementary Fig. 1.
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f1: Enrichment of functional EGFR-eYFP in the Sf21 microsomal fraction by repetitive cell-free synthesis.(a) Total protein yields after each cell-free reaction in the complete reaction mixture (R), the supernatant fraction (S) and the microsomal fraction (M). Error bars represent the standard deviation of triplicate analysis. (b) Autoradiography of corresponding samples after electrophoretic separation under denaturing conditions. (c) Yields of total protein and eYFP fluorescence after each cell-free reaction relative to the first reaction. (d) Confocal fluorescence image of EGFR-eYFP in microsomal fraction taken under hypoosmotic buffer conditions. (e) Western Blot of microsomal fractions with and without EGFR-eYFP after incubation in kinase buffer. Isotopic labeling was achieved by 14C-leucine supplementation. The western blots (e) have been adapted in contrast, brightness and sharpness for better visibility. The original images can be found in Supplementary Fig. 1.

Mentions: The synthesis of EGFR-eYFP based on the standard conditions previously described for the cell-free Sf21 system6 yielded relatively low amounts of several μg/ml within 90 minutes of reaction time (Fig. 1a). Although the successful synthesis of the EGFR-eYFP fusion protein was verified by different means (Fig. 1a–c), ligand-independent receptor activation based on autophosphorylation of Y1068 was not detectable (data not shown). Therefore, a batch of inherent Sf21 microsomes was repeatedly addressed by four consecutive cell-free reactions in order to enrich the cell-free synthesized EGFR-eYFP in the microsomal fraction and potentially promote ligand-independent receptor activation (Fig. 1a–c). As a result, an almost 3-fold increase of de novo synthesized EGFR was detected in the microsomal fraction yielding nearly 10 μg/ml of total protein (Fig. 1c,a, respectively). Autoradiography of isotopically labeled proteins revealed the predominant cell-free synthesis of two variants of the full-length receptor migrating at an apparent molecular weight slightly higher than the calculated 163 kDa (Fig. 1b). It should be noted that all cell-free reactions and assays presented within this study were performed in the presence of the caspase inhibitor Z-VAD-FMK to prevent degradation of cell-free synthesized EGFR, which was observed during a prolonged incubation in the reaction mixture in the absence of the inhibitor (Supplementary Fig. 2). Interestingly, the relative increase of fluorescence based on the eYFP fusion showed a higher increase with each additional cell-free reaction than was observed for the total protein (Fig. 1c). As expected, fluorescent spheres in the confocal image of the microsomal fraction taken under hypo-osmotic conditions after four consecutive cell-free reactions reflected the EGFR-eYFP fusion protein to be localized at the microsomal membranes, thereby supporting the hypothesis of membrane embedment due to a directed translocation mediated by the N-terminal melittin signal peptide in the cell-free environment (Fig. 1d). Finally, this enabled the detection of the Y1068 (corresponds to Y1090 in the synthesized construct including the melittin signal peptide) phosphorylation after incubation of the microsomal fraction in kinase buffer in the absence of ligand (Fig. 1e).


Cell-free synthesis of functional human epidermal growth factor receptor: Investigation of ligand-independent dimerization in Sf 21 microsomal membranes using non-canonical amino acids
Enrichment of functional EGFR-eYFP in the Sf21 microsomal fraction by repetitive cell-free synthesis.(a) Total protein yields after each cell-free reaction in the complete reaction mixture (R), the supernatant fraction (S) and the microsomal fraction (M). Error bars represent the standard deviation of triplicate analysis. (b) Autoradiography of corresponding samples after electrophoretic separation under denaturing conditions. (c) Yields of total protein and eYFP fluorescence after each cell-free reaction relative to the first reaction. (d) Confocal fluorescence image of EGFR-eYFP in microsomal fraction taken under hypoosmotic buffer conditions. (e) Western Blot of microsomal fractions with and without EGFR-eYFP after incubation in kinase buffer. Isotopic labeling was achieved by 14C-leucine supplementation. The western blots (e) have been adapted in contrast, brightness and sharpness for better visibility. The original images can be found in Supplementary Fig. 1.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC5037433&req=5

f1: Enrichment of functional EGFR-eYFP in the Sf21 microsomal fraction by repetitive cell-free synthesis.(a) Total protein yields after each cell-free reaction in the complete reaction mixture (R), the supernatant fraction (S) and the microsomal fraction (M). Error bars represent the standard deviation of triplicate analysis. (b) Autoradiography of corresponding samples after electrophoretic separation under denaturing conditions. (c) Yields of total protein and eYFP fluorescence after each cell-free reaction relative to the first reaction. (d) Confocal fluorescence image of EGFR-eYFP in microsomal fraction taken under hypoosmotic buffer conditions. (e) Western Blot of microsomal fractions with and without EGFR-eYFP after incubation in kinase buffer. Isotopic labeling was achieved by 14C-leucine supplementation. The western blots (e) have been adapted in contrast, brightness and sharpness for better visibility. The original images can be found in Supplementary Fig. 1.
Mentions: The synthesis of EGFR-eYFP based on the standard conditions previously described for the cell-free Sf21 system6 yielded relatively low amounts of several μg/ml within 90 minutes of reaction time (Fig. 1a). Although the successful synthesis of the EGFR-eYFP fusion protein was verified by different means (Fig. 1a–c), ligand-independent receptor activation based on autophosphorylation of Y1068 was not detectable (data not shown). Therefore, a batch of inherent Sf21 microsomes was repeatedly addressed by four consecutive cell-free reactions in order to enrich the cell-free synthesized EGFR-eYFP in the microsomal fraction and potentially promote ligand-independent receptor activation (Fig. 1a–c). As a result, an almost 3-fold increase of de novo synthesized EGFR was detected in the microsomal fraction yielding nearly 10 μg/ml of total protein (Fig. 1c,a, respectively). Autoradiography of isotopically labeled proteins revealed the predominant cell-free synthesis of two variants of the full-length receptor migrating at an apparent molecular weight slightly higher than the calculated 163 kDa (Fig. 1b). It should be noted that all cell-free reactions and assays presented within this study were performed in the presence of the caspase inhibitor Z-VAD-FMK to prevent degradation of cell-free synthesized EGFR, which was observed during a prolonged incubation in the reaction mixture in the absence of the inhibitor (Supplementary Fig. 2). Interestingly, the relative increase of fluorescence based on the eYFP fusion showed a higher increase with each additional cell-free reaction than was observed for the total protein (Fig. 1c). As expected, fluorescent spheres in the confocal image of the microsomal fraction taken under hypo-osmotic conditions after four consecutive cell-free reactions reflected the EGFR-eYFP fusion protein to be localized at the microsomal membranes, thereby supporting the hypothesis of membrane embedment due to a directed translocation mediated by the N-terminal melittin signal peptide in the cell-free environment (Fig. 1d). Finally, this enabled the detection of the Y1068 (corresponds to Y1090 in the synthesized construct including the melittin signal peptide) phosphorylation after incubation of the microsomal fraction in kinase buffer in the absence of ligand (Fig. 1e).

View Article: PubMed Central - PubMed

ABSTRACT

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.

No MeSH data available.


Related in: MedlinePlus