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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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Suppression efficiency of different EGFR-eYFP amber variants and photo-affinity cross-linking of receptors in Sf21 microsomal membranes.(a) Suppression efficiency of EGFR-eYFP templates with amber codons at positions 273, 285, 307, 687, 285 + 687 and vIII-deletion mutant at position 420 based on eYFP fluorescence from complete reaction mixture in relation to wild type and vIII template without amber codon. Error bars represent the standard deviation of triplicate analysis. (b) Autoradiography and (c) western blot of phosphotyrosine 1068 of selected constructs enriched in microsomal fraction by four consecutive cell-free reactions after photo-affinity cross-linking in kinase buffer. 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. 9.
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f4: Suppression efficiency of different EGFR-eYFP amber variants and photo-affinity cross-linking of receptors in Sf21 microsomal membranes.(a) Suppression efficiency of EGFR-eYFP templates with amber codons at positions 273, 285, 307, 687, 285 + 687 and vIII-deletion mutant at position 420 based on eYFP fluorescence from complete reaction mixture in relation to wild type and vIII template without amber codon. Error bars represent the standard deviation of triplicate analysis. (b) Autoradiography and (c) western blot of phosphotyrosine 1068 of selected constructs enriched in microsomal fraction by four consecutive cell-free reactions after photo-affinity cross-linking in kinase buffer. 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. 9.

Mentions: In order to investigate ligand-independent dimerization in the microsomal membranes, a set of amber mutants was generated to introduce AzF into selected positions in the extracellular (Amb285, Amb307) and intracellular domains (Amb687) of the cell-free synthesized EGFR-eYFP. Additionally, a double mutant (Amb285 + 687) and an amber variant of the constitutively active vIII deletion mutant of the EGFR (vIII-Amb420) were generated. First, the suppression efficiency of the different amber variants in the OcfTS was analyzed in relation to the synthesis of the corresponding receptor from templates without internal amber codon (Fig. 4a and Supplementary Fig. 8). All mutants were successfully synthesized but with varying efficiencies. The suppression efficiency decreased the more distant the amber codon was located downstream of the ATG start codon in the full-length EGFR-eYFP constructs. The extracellular mutants Amb285 and Amb307 exhibited more than 60% and the intracellular mutant Amb687 roughly 50% of suppression efficiency. The combination of two internal amber codons led to a further decrease of suppression efficiency down to 25%. This efficiency is in good accordance to previously published results, since suppression of multiple amber codons in general is reported to be very inefficient23. The amber variant of the vIII-deletion mutant Amb420 (corresponds to Amb687 in the full-length EGFR), which lacks 267 amino acids in the extracellular domain24, exhibited the highest suppression efficiency above 80%.


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
Suppression efficiency of different EGFR-eYFP amber variants and photo-affinity cross-linking of receptors in Sf21 microsomal membranes.(a) Suppression efficiency of EGFR-eYFP templates with amber codons at positions 273, 285, 307, 687, 285 + 687 and vIII-deletion mutant at position 420 based on eYFP fluorescence from complete reaction mixture in relation to wild type and vIII template without amber codon. Error bars represent the standard deviation of triplicate analysis. (b) Autoradiography and (c) western blot of phosphotyrosine 1068 of selected constructs enriched in microsomal fraction by four consecutive cell-free reactions after photo-affinity cross-linking in kinase buffer. 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. 9.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f4: Suppression efficiency of different EGFR-eYFP amber variants and photo-affinity cross-linking of receptors in Sf21 microsomal membranes.(a) Suppression efficiency of EGFR-eYFP templates with amber codons at positions 273, 285, 307, 687, 285 + 687 and vIII-deletion mutant at position 420 based on eYFP fluorescence from complete reaction mixture in relation to wild type and vIII template without amber codon. Error bars represent the standard deviation of triplicate analysis. (b) Autoradiography and (c) western blot of phosphotyrosine 1068 of selected constructs enriched in microsomal fraction by four consecutive cell-free reactions after photo-affinity cross-linking in kinase buffer. 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. 9.
Mentions: In order to investigate ligand-independent dimerization in the microsomal membranes, a set of amber mutants was generated to introduce AzF into selected positions in the extracellular (Amb285, Amb307) and intracellular domains (Amb687) of the cell-free synthesized EGFR-eYFP. Additionally, a double mutant (Amb285 + 687) and an amber variant of the constitutively active vIII deletion mutant of the EGFR (vIII-Amb420) were generated. First, the suppression efficiency of the different amber variants in the OcfTS was analyzed in relation to the synthesis of the corresponding receptor from templates without internal amber codon (Fig. 4a and Supplementary Fig. 8). All mutants were successfully synthesized but with varying efficiencies. The suppression efficiency decreased the more distant the amber codon was located downstream of the ATG start codon in the full-length EGFR-eYFP constructs. The extracellular mutants Amb285 and Amb307 exhibited more than 60% and the intracellular mutant Amb687 roughly 50% of suppression efficiency. The combination of two internal amber codons led to a further decrease of suppression efficiency down to 25%. This efficiency is in good accordance to previously published results, since suppression of multiple amber codons in general is reported to be very inefficient23. The amber variant of the vIII-deletion mutant Amb420 (corresponds to Amb687 in the full-length EGFR), which lacks 267 amino acids in the extracellular domain24, exhibited the highest suppression efficiency above 80%.

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