scholarly journals A Combined Cell-Free Protein Synthesis and Fluorescence-Based Approach to Investigate GPCR Binding Properties v1

2020 ◽  
Author(s):  
Anne Zemella ◽  
Theresa Richter ◽  
Lena Thoring ◽  
Stefan Kubick
Keyword(s):  
Protein Synthesis ◽  
Amino Acid ◽  
Membrane Proteins ◽  
Trna Synthetase ◽  
Fluorescent Labeling ◽  
Laser Scanning Microscopy ◽  
Free System ◽  
Free Protein ◽  
Canonical Amino Acid ◽  
Cell Free Protein Synthesis

Fluorescent labeling of de novo synthesized proteins is in particular a valuable tool for functional and structural studies of membrane proteins. In this context, we present two methods for the site-specific fluorescent labeling of difficult-to-express membrane proteins in combination with cell-free protein synthesis. The cell-free protein synthesis system is based on Chinese Hamster Ovary Cells (CHO) since this system contains endogenous membrane structures derived from the endoplasmic reticulum. These so-called microsomes enable a direct integration of membrane proteins into a biological membrane. In this protocol the first part describes the fluorescent labeling by using a precharged tRNA, loaded with a fluorescent amino acid. The second part describes the preparation of a modified aminoacyl-tRNA-synthetase and a suppressor tRNA that are applied to the CHO cell-free system to enable the incorporation of a non-canonical amino acid. The reactive group of the non-canonical amino acid is further coupled to a fluorescent dye. Both methods utilize the amber stop codon suppression technology. The successful fluorescent labeling of the model G protein-coupled receptor adenosine A2A (Adora2a) is analyzed by in-gel-fluorescence, a reporter protein assay, and confocal laser scanning microscopy (CLSM). Moreover, a ligand-dependent conformational change of the fluorescently labeled Adora2a was analyzed by bioluminescence resonance energy transfer (BRET).

scholarly journals Cell-Free Protein Synthesis v1

2020 ◽  
Author(s):  
Anne Zemella ◽  
Theresa Richter ◽  
Lena Thoring ◽  
Stefan Kubick
Keyword(s):  
Protein Synthesis ◽  
Amino Acid ◽  
Membrane Proteins ◽  
Trna Synthetase ◽  
Fluorescent Labeling ◽  
Laser Scanning Microscopy ◽  
Free System ◽  
Free Protein ◽  
Canonical Amino Acid ◽  
Cell Free Protein Synthesis

This is part 3.3 of the "A Combined Cell-Free Protein Synthesis and Fluorescence-Based Approach to Investigate GPCR Binding Properties" collection of protocols: https://www.protocols.io/view/a-combined-cell-free-protein-synthesis-and-fluores-bqntmven Collection Abstract: Fluorescent labeling of de novo synthesized proteins is in particular a valuable tool for functional and structural studies of membrane proteins. In this context, we present two methods for the site-specific fluorescent labeling of difficult-to-express membrane proteins in combination with cell-free protein synthesis. The cell-free protein synthesis system is based on Chinese Hamster Ovary Cells (CHO) since this system contains endogenous membrane structures derived from the endoplasmic reticulum. These so-called microsomes enable a direct integration of membrane proteins into a biological membrane. In this protocol the first part describes the fluorescent labeling by using a precharged tRNA, loaded with a fluorescent amino acid. The second part describes the preparation of a modified aminoacyl-tRNA-synthetase and a suppressor tRNA that are applied to the CHO cell-free system to enable the incorporation of a non-canonical amino acid. The reactive group of the non-canonical amino acid is further coupled to a fluorescent dye. Both methods utilize the amber stop codon suppression technology. The successful fluorescent labeling of the model G protein-coupled receptor adenosine A2A (Adora2a) is analyzed by in-gel-fluorescence, a reporter protein assay, and confocal laser scanning microscopy (CLSM). Moreover, a ligand-dependent conformational change of the fluorescently labeled Adora2a was analyzed by bioluminescence resonance energy transfer (BRET). For Introduction and Notes, please see: https://www.protocols.io/view/a-combined-cell-free-protein-synthesis-and-fluores-bqntmven/guidelines

scholarly journals Preparation of Enhanced Orthogonal Aminoacyl-tRNA-Synthetase v1

2020 ◽  
Author(s):  
Anne Zemella ◽  
Theresa Richter ◽  
Lena Thoring ◽  
Stefan Kubick
Keyword(s):  
Protein Synthesis ◽  
Amino Acid ◽  
Membrane Proteins ◽  
Trna Synthetase ◽  
Fluorescent Labeling ◽  
Laser Scanning Microscopy ◽  
Aminoacyl Trna Synthetase ◽  
Free Protein ◽  
Canonical Amino Acid ◽  
Cell Free Protein Synthesis

This is part 3.1 of the "A Combined Cell-Free Protein Synthesis and Fluorescence-Based Approach to Investigate GPCR Binding Properties" collection of protocols: https://www.protocols.io/view/a-combined-cell-free-protein-synthesis-and-fluores-bqntmven Collection Abstract: Fluorescent labeling of de novo synthesized proteins is in particular a valuable tool for functional and structural studies of membrane proteins. In this context, we present two methods for the site-specific fluorescent labeling of difficult-to-express membrane proteins in combination with cell-free protein synthesis. The cell-free protein synthesis system is based on Chinese Hamster Ovary Cells (CHO) since this system contains endogenous membrane structures derived from the endoplasmic reticulum. These so-called microsomes enable a direct integration of membrane proteins into a biological membrane. In this protocol the first part describes the fluorescent labeling by using a precharged tRNA, loaded with a fluorescent amino acid. The second part describes the preparation of a modified aminoacyl-tRNA-synthetase and a suppressor tRNA that are applied to the CHO cell-free system to enable the incorporation of a non-canonical amino acid. The reactive group of the non-canonical amino acid is further coupled to a fluorescent dye. Both methods utilize the amber stop codon suppression technology. The successful fluorescent labeling of the model G protein-coupled receptor adenosine A2A (Adora2a) is analyzed by in-gel-fluorescence, a reporter protein assay, and confocal laser scanning microscopy (CLSM). Moreover, a ligand-dependent conformational change of the fluorescently labeled Adora2a was analyzed by bioluminescence resonance energy transfer (BRET). For Introduction and Notes, please see: https://www.protocols.io/view/a-combined-cell-free-protein-synthesis-and-fluores-bqntmven/guidelines

scholarly journals Preparation of Suppressor tRNA v1

2020 ◽  
Author(s):  
Anne Zemella ◽  
Theresa Richter ◽  
Lena Thoring ◽  
Stefan Kubick
Keyword(s):  
Protein Synthesis ◽  
Amino Acid ◽  
Membrane Proteins ◽  
Trna Synthetase ◽  
Fluorescent Labeling ◽  
Laser Scanning Microscopy ◽  
Free Protein ◽  
Suppressor Trna ◽  
Canonical Amino Acid ◽  
Cell Free Protein Synthesis

This is part 3.2 of the "A Combined Cell-Free Protein Synthesis and Fluorescence-Based Approach to Investigate GPCR Binding Properties" collection of protocols: https://www.protocols.io/view/a-combined-cell-free-protein-synthesis-and-fluores-bqntmven Collection Abstract: Fluorescent labeling of de novo synthesized proteins is in particular a valuable tool for functional and structural studies of membrane proteins. In this context, we present two methods for the site-specific fluorescent labeling of difficult-to-express membrane proteins in combination with cell-free protein synthesis. The cell-free protein synthesis system is based on Chinese Hamster Ovary Cells (CHO) since this system contains endogenous membrane structures derived from the endoplasmic reticulum. These so-called microsomes enable a direct integration of membrane proteins into a biological membrane. In this protocol the first part describes the fluorescent labeling by using a precharged tRNA, loaded with a fluorescent amino acid. The second part describes the preparation of a modified aminoacyl-tRNA-synthetase and a suppressor tRNA that are applied to the CHO cell-free system to enable the incorporation of a non-canonical amino acid. The reactive group of the non-canonical amino acid is further coupled to a fluorescent dye. Both methods utilize the amber stop codon suppression technology. The successful fluorescent labeling of the model G protein-coupled receptor adenosine A2A (Adora2a) is analyzed by in-gel-fluorescence, a reporter protein assay, and confocal laser scanning microscopy (CLSM). Moreover, a ligand-dependent conformational change of the fluorescently labeled Adora2a was analyzed by bioluminescence resonance energy transfer (BRET). For Introduction and Notes, please see: https://www.protocols.io/view/a-combined-cell-free-protein-synthesis-and-fluores-bqntmven/guidelines

scholarly journals Analysis of De Novo Synthesized Proteins v1

2020 ◽  
Author(s):  
Anne Zemella ◽  
Theresa Richter ◽  
Lena Thoring ◽  
Stefan Kubick
Keyword(s):  
Protein Synthesis ◽  
Amino Acid ◽  
Membrane Proteins ◽  
De Novo ◽  
Trna Synthetase ◽  
Fluorescent Labeling ◽  
Laser Scanning Microscopy ◽  
Free Protein ◽  
Canonical Amino Acid ◽  
Cell Free Protein Synthesis

This is part 3.4 of the "A Combined Cell-Free Protein Synthesis and Fluorescence-Based Approach to Investigate GPCR Binding Properties" collection of protocols: https://www.protocols.io/view/a-combined-cell-free-protein-synthesis-and-fluores-bqntmven Collection Abstract: Fluorescent labeling of de novo synthesized proteins is in particular a valuable tool for functional and structural studies of membrane proteins. In this context, we present two methods for the site-specific fluorescent labeling of difficult-to-express membrane proteins in combination with cell-free protein synthesis. The cell-free protein synthesis system is based on Chinese Hamster Ovary Cells (CHO) since this system contains endogenous membrane structures derived from the endoplasmic reticulum. These so-called microsomes enable a direct integration of membrane proteins into a biological membrane. In this protocol the first part describes the fluorescent labeling by using a precharged tRNA, loaded with a fluorescent amino acid. The second part describes the preparation of a modified aminoacyl-tRNA-synthetase and a suppressor tRNA that are applied to the CHO cell-free system to enable the incorporation of a non-canonical amino acid. The reactive group of the non-canonical amino acid is further coupled to a fluorescent dye. Both methods utilize the amber stop codon suppression technology. The successful fluorescent labeling of the model G protein-coupled receptor adenosine A2A (Adora2a) is analyzed by in-gel-fluorescence, a reporter protein assay, and confocal laser scanning microscopy (CLSM). Moreover, a ligand-dependent conformational change of the fluorescently labeled Adora2a was analyzed by bioluminescence resonance energy transfer (BRET). For Introduction and Notes, please see: https://www.protocols.io/view/a-combined-cell-free-protein-synthesis-and-fluores-bqntmven/guidelines

scholarly journals Cell-Free Protein Synthesis Using S30 Extracts from Escherichia coli RFzero Strains for Efficient Incorporation of Non-Natural Amino Acids into Proteins

2019 ◽  
Vol 20 (3) ◽  
pp. 492 ◽  
Author(s):  
Jiro Adachi ◽  
Kazushige Katsura ◽  
Eiko Seki ◽  
Chie Takemoto ◽  
Mikako Shirouzu ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Amino Acids ◽  
Protein Synthesis ◽  
Trna Synthetase ◽  
Translation Efficiency ◽  
Free System ◽  
Free Protein ◽  
Cell Free System ◽  
Natural Amino Acids ◽  
Cell Free Protein Synthesis

Cell-free protein synthesis is useful for synthesizing difficult targets. The site-specific incorporation of non-natural amino acids into proteins is a powerful protein engineering method. In this study, we optimized the protocol for cell extract preparation from the Escherichia coli strain RFzero-iy, which is engineered to lack release factor 1 (RF-1). The BL21(DE3)-based RFzero-iy strain exhibited quite high cell-free protein productivity, and thus we established the protocols for its cell culture and extract preparation. In the presence of 3-iodo-l-tyrosine (IY), cell-free protein synthesis using the RFzero-iy-based S30 extract translated the UAG codon to IY at various sites with a high translation efficiency of >90%. In the absence of IY, the RFzero-iy-based cell-free system did not translate UAG to any amino acid, leaving UAG unassigned. Actually, UAG was readily reassigned to various non-natural amino acids, by supplementing them with their specific aminoacyl-tRNA synthetase variants (and their specific tRNAs) into the system. The high incorporation rate of our RFzero-iy-based cell-free system enables the incorporation of a variety of non-natural amino acids into multiple sites of proteins. The present strategy to create the RFzero strain is rapid, and thus promising for RF-1 deletions of various E. coli strains genomically engineered for specific requirements.

Amino acid stabilization for cell-free protein synthesis by modification of the Escherichia coli genome

2004 ◽  
Vol 6 (3) ◽  
pp. 197-203 ◽  
Author(s):  
Nathalie Michel-Reydellet ◽  
Kara Calhoun ◽  
James Swartz
Keyword(s):  
Escherichia Coli ◽  
Protein Synthesis ◽  
Amino Acid ◽  
Free Protein ◽  
Cell Free Protein Synthesis

Advances in cell-free protein synthesis for the functional and structural analysis of membrane proteins

2011 ◽  
Vol 28 (3) ◽  
pp. 262-271 ◽  
Author(s):  
Friederike Junge ◽  
Stefan Haberstock ◽  
Christian Roos ◽  
Susanne Stefer ◽  
Davide Proverbio ◽  
...  
Keyword(s):  
Protein Synthesis ◽  
Structural Analysis ◽  
Membrane Proteins ◽  
Free Protein ◽  
Cell Free Protein Synthesis
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