scholarly journals Evaluation of cell‐free protein synthesis for the crystallization of membrane proteins – a case study on a member of the glutamate transporter family fromStaphylothermus marinus

FEBS Journal ◽  
2013 ◽  
Vol 280 (4) ◽  
pp. 1112-1125 ◽  
Author(s):  
Michael Jaehme ◽  
Hartmut Michel
Keyword(s):  
Protein Synthesis ◽  
Membrane Proteins ◽  
Glutamate Transporter ◽  
Free Protein ◽  
Transporter Family ◽  
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

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 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

Expression screening of membrane proteins with cell-free protein synthesis

2012 ◽  
Vol 82 (1) ◽  
pp. 218-225 ◽  
Author(s):  
Linnéa Isaksson ◽  
Johan Enberg ◽  
Richard Neutze ◽  
B. Göran Karlsson ◽  
Anders Pedersen
Keyword(s):  
Protein Synthesis ◽  
Membrane Proteins ◽  
Free Protein ◽  
Expression Screening ◽  
Cell Free Protein Synthesis

scholarly journals Production of Membrane Proteins Using Cell-free Protein Synthesis

2011 ◽  
Vol 51 (3) ◽  
pp. 128-129
Author(s):  
Kazumi SHIMONO ◽  
Mikako SHIROUZU ◽  
Shigeyuki YOKOYAMA
Keyword(s):  
Protein Synthesis ◽  
Membrane Proteins ◽  
Free Protein ◽  
Cell Free Protein Synthesis

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

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