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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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IRES-mediated cell-free synthesis of EGFR-eYFP-AzF687 by amber suppression using an orthogonal tRNA/synthetase pair and subsequent chemo-selective fluorescence modification.(a) eYFP fluorescence obtained using the standard template (−IRES) and the IRES template with an amber codon at position 687 (+IRES) in the absence (−PG) and the presence of poly G (+PG). Error bars represent the standard deviation of triplicate analysis. (b) Autoradiography and (c) in-gel fluorescence of microsomal fractions from reactions performed using the IRES template without (WT) and with amber codon (Amb) in the presence of poly G after treatment with DyLight650 phosphine. Isotopic labeling was achieved by 14C-leucine supplementation. NTC: control reaction without DNA template.
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f3: IRES-mediated cell-free synthesis of EGFR-eYFP-AzF687 by amber suppression using an orthogonal tRNA/synthetase pair and subsequent chemo-selective fluorescence modification.(a) eYFP fluorescence obtained using the standard template (−IRES) and the IRES template with an amber codon at position 687 (+IRES) in the absence (−PG) and the presence of poly G (+PG). Error bars represent the standard deviation of triplicate analysis. (b) Autoradiography and (c) in-gel fluorescence of microsomal fractions from reactions performed using the IRES template without (WT) and with amber codon (Amb) in the presence of poly G after treatment with DyLight650 phosphine. Isotopic labeling was achieved by 14C-leucine supplementation. NTC: control reaction without DNA template.

Mentions: With the aim to further increase the yields of full-length suppression product, we investigated the influence of IRES-mediated synthesis and poly G in the coupled OcfTS. For this purpose, amber codons were introduced into the EGFR-eYFP templates with and without IRES, at the position corresponding to amino acid 687 in the translated receptor (corresponds to V665 in the wild type receptor without signal peptide) and synthesis of the full-length suppression product was measured based on fluorescence of the eYFP moiety only being translated upon successful amber suppression. Without additional Mg2+, which was previously described to enhance the productivity of the OcfTS6, only a small amount of suppression product was synthesized using standard conditions. The suppression product was predominantly found in the supernatant fraction (Fig. 3a, −IRES, −PG). Using the IRES template and adjusted buffer conditions7 with elevated levels of K+ and Mg2+, yields increased in the microsomal fraction in the absence of poly G (Fig. 3a, +IRES, −PG). According to the findings for the wild type protein, highest yields were obtained using the IRES template and supplementing the cell-free reaction with poly G (Fig. 3a, +IRES, +PG). In accordance with the results obtained for the wild type EGFR-eYFP (Fig. 2b), the increase in the supernatant fraction was higher than in the microsomal fraction (Fig. 3a). Total yields of the suppression product, estimated from corresponding yields of the wild type protein, were 4.47 μg/ml in the supernatant fraction and 2.66 μg/ml in the microsomal fraction (Table 1, Amb). The incorporation of AzF, using the adapted conditions, was verified by selective Staudinger ligation of cell-free synthesized receptors in the microsomal fraction with the fluorescent phosphine dye (Fig. 3c, Amb). The specificity of incorporation was found to be good, as no fluorescence modification was detected for the wild type control (Fig. 3c, WT).


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
IRES-mediated cell-free synthesis of EGFR-eYFP-AzF687 by amber suppression using an orthogonal tRNA/synthetase pair and subsequent chemo-selective fluorescence modification.(a) eYFP fluorescence obtained using the standard template (−IRES) and the IRES template with an amber codon at position 687 (+IRES) in the absence (−PG) and the presence of poly G (+PG). Error bars represent the standard deviation of triplicate analysis. (b) Autoradiography and (c) in-gel fluorescence of microsomal fractions from reactions performed using the IRES template without (WT) and with amber codon (Amb) in the presence of poly G after treatment with DyLight650 phosphine. Isotopic labeling was achieved by 14C-leucine supplementation. NTC: control reaction without DNA template.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f3: IRES-mediated cell-free synthesis of EGFR-eYFP-AzF687 by amber suppression using an orthogonal tRNA/synthetase pair and subsequent chemo-selective fluorescence modification.(a) eYFP fluorescence obtained using the standard template (−IRES) and the IRES template with an amber codon at position 687 (+IRES) in the absence (−PG) and the presence of poly G (+PG). Error bars represent the standard deviation of triplicate analysis. (b) Autoradiography and (c) in-gel fluorescence of microsomal fractions from reactions performed using the IRES template without (WT) and with amber codon (Amb) in the presence of poly G after treatment with DyLight650 phosphine. Isotopic labeling was achieved by 14C-leucine supplementation. NTC: control reaction without DNA template.
Mentions: With the aim to further increase the yields of full-length suppression product, we investigated the influence of IRES-mediated synthesis and poly G in the coupled OcfTS. For this purpose, amber codons were introduced into the EGFR-eYFP templates with and without IRES, at the position corresponding to amino acid 687 in the translated receptor (corresponds to V665 in the wild type receptor without signal peptide) and synthesis of the full-length suppression product was measured based on fluorescence of the eYFP moiety only being translated upon successful amber suppression. Without additional Mg2+, which was previously described to enhance the productivity of the OcfTS6, only a small amount of suppression product was synthesized using standard conditions. The suppression product was predominantly found in the supernatant fraction (Fig. 3a, −IRES, −PG). Using the IRES template and adjusted buffer conditions7 with elevated levels of K+ and Mg2+, yields increased in the microsomal fraction in the absence of poly G (Fig. 3a, +IRES, −PG). According to the findings for the wild type protein, highest yields were obtained using the IRES template and supplementing the cell-free reaction with poly G (Fig. 3a, +IRES, +PG). In accordance with the results obtained for the wild type EGFR-eYFP (Fig. 2b), the increase in the supernatant fraction was higher than in the microsomal fraction (Fig. 3a). Total yields of the suppression product, estimated from corresponding yields of the wild type protein, were 4.47 μg/ml in the supernatant fraction and 2.66 μg/ml in the microsomal fraction (Table 1, Amb). The incorporation of AzF, using the adapted conditions, was verified by selective Staudinger ligation of cell-free synthesized receptors in the microsomal fraction with the fluorescent phosphine dye (Fig. 3c, Amb). The specificity of incorporation was found to be good, as no fluorescence modification was detected for the wild type control (Fig. 3c, WT).

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