Preparation of Suppressor tRNA
Lena Thoring, Stefan Kubick, Anne Zemella, Theresa Richter
Cell-free protein synthesis
G protein-coupled receptor
Protein modification
Non-canonical amino acids
Amber suppression
Confocal laser scanning microscopy
Abstract
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 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
Steps
3.2.1 Generation of PCR Product
For specific and homogenous 3’-ends of the suppressor tRNA, an additional PCR step before transcription reaction is included. Therefore, the reverse primer contains a 2’-OMe-group to prevent unspecific nucleotides at the 3’-end of the tRNA that can be added by the T7 polymerase during transcription reaction. Amplify the template by pipetting in a PCR tube final concentrations of 1x, 0.2millimolar (mM), 0.5micromolar (µM), 0.5micromolar (µM), 2.5millimolar (mM), 0.01ng/μl and 0.04U/μl. Fill the reaction with water to a final volume of 250µL ( see Note 5 ).
3.2.2 Generation of RNA Transcript
Thaw the components for in vitro transcription On ice and pipette the reaction at Room temperature. Mix 1x, 1x, 1U/μl and 8ng/μl
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Fill the reaction with water to the final volume of 500µL.
Incubate the reaction for 3h 0m 0s–6h 0m 0s at 500rpm.
Centrifuge the RNA at 12000x g und use the supernatant for the DNAseI treatment ( see Note 6 ).
Add 1U per 1 μg DNA .
Incubate for 500rpm.
3.2.3 RNA Isolation and Folding
Incubate for 0h 5m 0s at Room temperature.
Add 200µL for 1mL and mix carefully for 0h 0m 15s by inverting.
Incubate for 0h 3m 0s at Room temperature.
Centrifuge at 12000x g,4°C. Isolate the aqueous phase ( see Note 7 ).
Add 500µL for 1mL and mix carefully.
Incubate 0h 3m 0s at 4°C.
Centrifuge at 15000x g,0h 0m 0s at least for 1h 0m 0s at 4°C and discard the supernatant.
Overlay the pellet with 1mL for 1mL and incubate for 0h 30m 0s at -20°C.
Centrifuge at 7500x g,4°C. Discard the supernatant and air dry the pellet.
Solve the pellet in water. Measure concentration using a NanoDrop and adjust the concentration to 100micromolar (µM).
Fold the tRNA by slowly decreasing the temperature from 80°C to 25°C in a PCR cycler. The tRNA can be stored at -80°C after shock freezing in liquid nitrogen.
