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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 N-glycosylated EGFR-eYFP and its embedment in Sf21 microsomal membranes.(a) Schematic representation of standard (top) and IRES template (bottom) including regulatory elements. (b) Yields of total protein and eYFP fluorescence obtained using the standard (−IRES) or IRES template (+IRES) in the absence (−PG) or presence of poly G (+PG) relative to the standard reaction (−IRES, −PG). Error bars represent the standard deviation of triplicate analysis. (c–e) Autoradiography of IRES-mediated synthesis reactions in the presence of poly G after electrophoretic separation. c) Deglycosylation assay of complete reaction mixtures with Endo H or PNGase F. (d,e) Protease protection assay of microsomal fractions with Proteinase K in the absence (−Detergent) or presence of Triton X-100 (+Detergent). Isotopic labeling was achieved by 14C-leucine supplementation.
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f2: IRES-mediated cell-free synthesis of N-glycosylated EGFR-eYFP and its embedment in Sf21 microsomal membranes.(a) Schematic representation of standard (top) and IRES template (bottom) including regulatory elements. (b) Yields of total protein and eYFP fluorescence obtained using the standard (−IRES) or IRES template (+IRES) in the absence (−PG) or presence of poly G (+PG) relative to the standard reaction (−IRES, −PG). Error bars represent the standard deviation of triplicate analysis. (c–e) Autoradiography of IRES-mediated synthesis reactions in the presence of poly G after electrophoretic separation. c) Deglycosylation assay of complete reaction mixtures with Endo H or PNGase F. (d,e) Protease protection assay of microsomal fractions with Proteinase K in the absence (−Detergent) or presence of Triton X-100 (+Detergent). Isotopic labeling was achieved by 14C-leucine supplementation.

Mentions: Although the repetitive synthesis enabled assessment of the functionality of cell-free synthesized EGFR-eYFP, the underlying methodology is comparatively laborious as well as time and material consuming. Each reaction needs to be individually constituted using fresh components followed by consecutive incubation steps. Thus, in order to increase the yield in a single batch reaction, the influence of the CrPV-IRES was investigated, which has been described to provide an enhanced synthesis rate in different eukaryotic cell-free systems due to protein translation proceeding independent of initiation factors7. Additionally, the supplementation with poly G was investigated, which has been reported to inhibit RNase activity in a cell-free wheat germ system20. The CrPV-IRES was implemented into the corresponding vector upstream of the EGFR-eYFP gene with the ATG start codon of the melittin signal sequence substituted by GCT (Fig. 2a). In this way, total protein yields were increased in the supernatant as well as in the microsomal fraction. An additional increase in protein yield was observed in the presence of poly G (Fig. 2b and Table 1, WT). The increase in total protein yield was slightly higher than the increase in fluorescence of the eYFP fusion protein. IRES-mediated synthesis in the presence of poly G provided the highest increase with 12.8 μg/ml in the supernatant fraction and 8 μg/ml in the microsomal fraction, whereas supplementation with poly G completely inhibited cell-free protein synthesis using the DNA template without IRES (data not shown). The reaction lifetime was not prolonged and remained at 90 to 120 minutes according to the reaction lifetime of the standard reaction without IRES (Supplementary Fig. 3). Protein integrity of IRES-mediated cell-free synthesized EGFR-eYFP in the presence of poly G was verified by SDS-PAGE followed by autoradiography. Treatment of reaction aliquots with PNGase F and Endo H revealed the before-mentioned two protein variants representing the N-glycosylated as well as non-glycosylated form of the receptor (Fig. 2c).


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 N-glycosylated EGFR-eYFP and its embedment in Sf21 microsomal membranes.(a) Schematic representation of standard (top) and IRES template (bottom) including regulatory elements. (b) Yields of total protein and eYFP fluorescence obtained using the standard (−IRES) or IRES template (+IRES) in the absence (−PG) or presence of poly G (+PG) relative to the standard reaction (−IRES, −PG). Error bars represent the standard deviation of triplicate analysis. (c–e) Autoradiography of IRES-mediated synthesis reactions in the presence of poly G after electrophoretic separation. c) Deglycosylation assay of complete reaction mixtures with Endo H or PNGase F. (d,e) Protease protection assay of microsomal fractions with Proteinase K in the absence (−Detergent) or presence of Triton X-100 (+Detergent). Isotopic labeling was achieved by 14C-leucine supplementation.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f2: IRES-mediated cell-free synthesis of N-glycosylated EGFR-eYFP and its embedment in Sf21 microsomal membranes.(a) Schematic representation of standard (top) and IRES template (bottom) including regulatory elements. (b) Yields of total protein and eYFP fluorescence obtained using the standard (−IRES) or IRES template (+IRES) in the absence (−PG) or presence of poly G (+PG) relative to the standard reaction (−IRES, −PG). Error bars represent the standard deviation of triplicate analysis. (c–e) Autoradiography of IRES-mediated synthesis reactions in the presence of poly G after electrophoretic separation. c) Deglycosylation assay of complete reaction mixtures with Endo H or PNGase F. (d,e) Protease protection assay of microsomal fractions with Proteinase K in the absence (−Detergent) or presence of Triton X-100 (+Detergent). Isotopic labeling was achieved by 14C-leucine supplementation.
Mentions: Although the repetitive synthesis enabled assessment of the functionality of cell-free synthesized EGFR-eYFP, the underlying methodology is comparatively laborious as well as time and material consuming. Each reaction needs to be individually constituted using fresh components followed by consecutive incubation steps. Thus, in order to increase the yield in a single batch reaction, the influence of the CrPV-IRES was investigated, which has been described to provide an enhanced synthesis rate in different eukaryotic cell-free systems due to protein translation proceeding independent of initiation factors7. Additionally, the supplementation with poly G was investigated, which has been reported to inhibit RNase activity in a cell-free wheat germ system20. The CrPV-IRES was implemented into the corresponding vector upstream of the EGFR-eYFP gene with the ATG start codon of the melittin signal sequence substituted by GCT (Fig. 2a). In this way, total protein yields were increased in the supernatant as well as in the microsomal fraction. An additional increase in protein yield was observed in the presence of poly G (Fig. 2b and Table 1, WT). The increase in total protein yield was slightly higher than the increase in fluorescence of the eYFP fusion protein. IRES-mediated synthesis in the presence of poly G provided the highest increase with 12.8 μg/ml in the supernatant fraction and 8 μg/ml in the microsomal fraction, whereas supplementation with poly G completely inhibited cell-free protein synthesis using the DNA template without IRES (data not shown). The reaction lifetime was not prolonged and remained at 90 to 120 minutes according to the reaction lifetime of the standard reaction without IRES (Supplementary Fig. 3). Protein integrity of IRES-mediated cell-free synthesized EGFR-eYFP in the presence of poly G was verified by SDS-PAGE followed by autoradiography. Treatment of reaction aliquots with PNGase F and Endo H revealed the before-mentioned two protein variants representing the N-glycosylated as well as non-glycosylated form of the receptor (Fig. 2c).

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