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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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Selective cross-linking of receptors with incorporated AzF by strain-promoted cycloaddition using the bis-COMBO linker.(a) Schematic representation of cross-linking. Receptor- (blue) eYFP (yellow) fusion proteins are embedded in the membranes with incorporated AzF (N3) on the outside of the Sf21 microsomal membranes. Treatment of the microsomal fractions with the bis-COMBO linker in kinase buffer in the absence of ATP leads to strain-promoted cycloaddition to form covalently linked receptor dimers. (b) Autoradiography and (c) western blot of phosphotyrosine 1068 of selected constructs enriched in microsomal fractions by four consecutive cell-free reactions, after treatment with bis-COMBO linker. Following the cross-linking samples were incubated in kinase buffer in the absence (−) and presence of ATP (+) to allow for autophosphorylation. Isotopic labeling was achieved by 14C-leucine supplementation. Asterisks mark the covalently cross-linked dimers. The autoradiogram (b) and western blot (c) have been adapted in contrast, brightness and sharpness for better visibility. The original images can be found in Supplementary Fig. 10.
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f5: Selective cross-linking of receptors with incorporated AzF by strain-promoted cycloaddition using the bis-COMBO linker.(a) Schematic representation of cross-linking. Receptor- (blue) eYFP (yellow) fusion proteins are embedded in the membranes with incorporated AzF (N3) on the outside of the Sf21 microsomal membranes. Treatment of the microsomal fractions with the bis-COMBO linker in kinase buffer in the absence of ATP leads to strain-promoted cycloaddition to form covalently linked receptor dimers. (b) Autoradiography and (c) western blot of phosphotyrosine 1068 of selected constructs enriched in microsomal fractions by four consecutive cell-free reactions, after treatment with bis-COMBO linker. Following the cross-linking samples were incubated in kinase buffer in the absence (−) and presence of ATP (+) to allow for autophosphorylation. Isotopic labeling was achieved by 14C-leucine supplementation. Asterisks mark the covalently cross-linked dimers. The autoradiogram (b) and western blot (c) have been adapted in contrast, brightness and sharpness for better visibility. The original images can be found in Supplementary Fig. 10.

Mentions: Finally, the two mutants that incorporate AzF in the intracellular juxtamembrane domain were used to exploit a novel cross-linking agent composed of two COMBO groups connected by a tetraethylene glycol linker in order to form stable, covalently linked receptor dimers (Fig. 5a). For that purpose, the EGFR-eYFP-AzF687 and the vIII-AzF420 mutants were enriched in the microsomal fractions by four consecutive cell-free reactions. Both proteins were subsequently treated with the bis-COMBO linker in kinase buffer in the absence of ATP. Following the cross-linking, aliquots were subjected to the tyrosine kinase assay to analyze the phosphorylation of covalently linked dimers. Autoradiography of isotopically labeled proteins revealed weak but distinct proteins migrating at approximately the molecular weight corresponding to receptor dimers (Fig. 5b). In contrast, the controls synthesized from the wild type constructs without internal amber codon were not cross-linked, underlining the specificity of the reaction between the incorporated azides and the COMBO groups. The amount of cross-linked dimers, estimated from the autoradiogram, was approximately 4% for EGFR-eYFP-AzF687 and 10% for vIII-AzF420 (Supplementary Fig. 11). Further, the phosphorylation of Y1068 was found to occur even in the pre-linked dimers, when incubated in kinase buffer in the presence of ATP (Fig. 5c).


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
Selective cross-linking of receptors with incorporated AzF by strain-promoted cycloaddition using the bis-COMBO linker.(a) Schematic representation of cross-linking. Receptor- (blue) eYFP (yellow) fusion proteins are embedded in the membranes with incorporated AzF (N3) on the outside of the Sf21 microsomal membranes. Treatment of the microsomal fractions with the bis-COMBO linker in kinase buffer in the absence of ATP leads to strain-promoted cycloaddition to form covalently linked receptor dimers. (b) Autoradiography and (c) western blot of phosphotyrosine 1068 of selected constructs enriched in microsomal fractions by four consecutive cell-free reactions, after treatment with bis-COMBO linker. Following the cross-linking samples were incubated in kinase buffer in the absence (−) and presence of ATP (+) to allow for autophosphorylation. Isotopic labeling was achieved by 14C-leucine supplementation. Asterisks mark the covalently cross-linked dimers. The autoradiogram (b) and western blot (c) have been adapted in contrast, brightness and sharpness for better visibility. The original images can be found in Supplementary Fig. 10.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f5: Selective cross-linking of receptors with incorporated AzF by strain-promoted cycloaddition using the bis-COMBO linker.(a) Schematic representation of cross-linking. Receptor- (blue) eYFP (yellow) fusion proteins are embedded in the membranes with incorporated AzF (N3) on the outside of the Sf21 microsomal membranes. Treatment of the microsomal fractions with the bis-COMBO linker in kinase buffer in the absence of ATP leads to strain-promoted cycloaddition to form covalently linked receptor dimers. (b) Autoradiography and (c) western blot of phosphotyrosine 1068 of selected constructs enriched in microsomal fractions by four consecutive cell-free reactions, after treatment with bis-COMBO linker. Following the cross-linking samples were incubated in kinase buffer in the absence (−) and presence of ATP (+) to allow for autophosphorylation. Isotopic labeling was achieved by 14C-leucine supplementation. Asterisks mark the covalently cross-linked dimers. The autoradiogram (b) and western blot (c) have been adapted in contrast, brightness and sharpness for better visibility. The original images can be found in Supplementary Fig. 10.
Mentions: Finally, the two mutants that incorporate AzF in the intracellular juxtamembrane domain were used to exploit a novel cross-linking agent composed of two COMBO groups connected by a tetraethylene glycol linker in order to form stable, covalently linked receptor dimers (Fig. 5a). For that purpose, the EGFR-eYFP-AzF687 and the vIII-AzF420 mutants were enriched in the microsomal fractions by four consecutive cell-free reactions. Both proteins were subsequently treated with the bis-COMBO linker in kinase buffer in the absence of ATP. Following the cross-linking, aliquots were subjected to the tyrosine kinase assay to analyze the phosphorylation of covalently linked dimers. Autoradiography of isotopically labeled proteins revealed weak but distinct proteins migrating at approximately the molecular weight corresponding to receptor dimers (Fig. 5b). In contrast, the controls synthesized from the wild type constructs without internal amber codon were not cross-linked, underlining the specificity of the reaction between the incorporated azides and the COMBO groups. The amount of cross-linked dimers, estimated from the autoradiogram, was approximately 4% for EGFR-eYFP-AzF687 and 10% for vIII-AzF420 (Supplementary Fig. 11). Further, the phosphorylation of Y1068 was found to occur even in the pre-linked dimers, when incubated in kinase buffer in the presence of ATP (Fig. 5c).

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