Rapid One-Step Capturing of Native, Cell-Free Synthesized and Membrane-Embedded GLP-1R
Abstract
:1. Introduction
2. Results
2.1. Cell-Free Synthesis of Glycosylated GLP-1R in Eukaryotic Lysates
2.2. Site-Specific and Co-Translational Biotinylation of a GLP-1R Amber Mutant Using Precharged tRNAs
2.3. Immobilization of Biotinylated GLP-1R-L260amb onto Magnetic Beads
2.4. Analysis of Microsomal Membrane-Embedded and Bead-Captured GLP-1R by Confocal Laser Scanning Microscopy (CLSM)
2.5. Analyzing the Accessibility of Bead-Captured GLP-1R ECD for Antibody Binding
2.6. Binding of Exendin-4 Peptide Ligand to Bead-Captured GLP-1R Preparations
3. Discussion
4. Materials and Methods
4.1. DNA Template Generation
4.2. Cell-Free Synthesis in Eukaryotic Lysates and Introduction of Non-Canonical Amino Acids into GLP-1R
4.3. Quantitative and Qualitative Analysis of 14C-leucine-Labeled Proteins
4.4. Silver Staining
4.5. Glycosylation Analysis
4.6. Protein Purification and Immobilization on Magnetic Beads
4.7. Analysis of Protein Translocation Using Confocal Laser Scanning Microscopy
4.8. Bead-Based ELISA
4.9. Ligand-Binding Assays
Supplementary Materials
Author Contributions
Funding
Institutional Review Board Statement
Informed Consent Statement
Data Availability Statement
Acknowledgments
Conflicts of Interest
References
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Haueis, L.; Stech, M.; Schneider, E.; Lanz, T.; Hebel, N.; Zemella, A.; Kubick, S. Rapid One-Step Capturing of Native, Cell-Free Synthesized and Membrane-Embedded GLP-1R. Int. J. Mol. Sci. 2023, 24, 2808. https://doi.org/10.3390/ijms24032808
Haueis L, Stech M, Schneider E, Lanz T, Hebel N, Zemella A, Kubick S. Rapid One-Step Capturing of Native, Cell-Free Synthesized and Membrane-Embedded GLP-1R. International Journal of Molecular Sciences. 2023; 24(3):2808. https://doi.org/10.3390/ijms24032808
Chicago/Turabian StyleHaueis, Lisa, Marlitt Stech, Eberhard Schneider, Thorsten Lanz, Nicole Hebel, Anne Zemella, and Stefan Kubick. 2023. "Rapid One-Step Capturing of Native, Cell-Free Synthesized and Membrane-Embedded GLP-1R" International Journal of Molecular Sciences 24, no. 3: 2808. https://doi.org/10.3390/ijms24032808
APA StyleHaueis, L., Stech, M., Schneider, E., Lanz, T., Hebel, N., Zemella, A., & Kubick, S. (2023). Rapid One-Step Capturing of Native, Cell-Free Synthesized and Membrane-Embedded GLP-1R. International Journal of Molecular Sciences, 24(3), 2808. https://doi.org/10.3390/ijms24032808