TITLE: Preparation of Suppressor tRNA
AUTHORS: Anne Zemella, Theresa Richter, Lena Thoring, Stefan Kubick

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><span style = "font-weight:bold;">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</span><span style = "font-weight:bold;font-weight:bold;">:</span><span style = ":;font-weight:bold;"> </span><a href="https://www.protocols.io/view/a-combined-cell-free-protein-synthesis-and-fluores-bqntmven" style = "text-decoration:underline;color:blue;cursor:pointer;"><span style = ":;font-weight:bold;">https://www.protocols.io/view/a-combined-cell-free-protein-synthesis-and-fluores-bqntmve</span></a><a href="https://www.protocols.io/view/a-combined-cell-free-protein-synthesis-and-fluores-bqntmven/guidelines" style = "text-decoration:underline;color:blue;cursor:pointer;"><span style = ":;:;font-weight:bold;">n</span></a></div><br/><div class = "text-block"><span style = "font-weight:bold;">Collection Abstract: </span><span>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).</span></div><br/><div class = "text-block"><span>For </span><span style = "font-weight:bold;">Introduction and Notes</span><span>, please see: </span><a href="https://www.protocols.io/view/a-combined-cell-free-protein-synthesis-and-fluores-bqntmven/guidelines" style = "text-decoration:underline;color:blue;cursor:pointer;"><span style = ":;">https://www.protocols.io/view/a-combined-cell-free-protein-synthesis-and-fluores-bqntmven/guidelines</span></a></div></div>

[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 , , , , ,  and . Fill the reaction with water to a final volume of  (see Note 5).
[Taq Buffer]
[dNTP mix]
[forward primer]
[reverse primer]
[MgCl2]
[plasmid]
[Taq DNA polymerase]
250 µl
?. [3.2.2 Generation of RNA Transcript]
Thaw the components for in vitro transcription  and pipette the reaction at . Mix , ,  and .
on ice
0 Room temperature
[transcription buffer]
[NTP mix]
[T7 RNA Polymerase]
[template DNA]
?. [3.2.2 Generation of RNA Transcript]
Centrifuge the RNA at  und use the supernatant for the DNAseI treatment (see Note 6).
Centrifuge: 12000 34, 1 min
?. [3.2.3 RNA Isolation and Folding]
Add a threefold volume of TRIzol to the transcription reaction and mix carefully.
Handle the TRIzol and chloroform reagent with care and use a fume hood.
?. [3.2.3 RNA Isolation and Folding]
Centrifuge at  at least for  at  and discard the supernatant.
Centrifuge: 15000 34
4 °C
?. [3.2.3 RNA Isolation and Folding]
Fold the tRNA by slowly decreasing the temperature from  to  in a PCR cycler. The tRNA can be stored at  after shock freezing in liquid nitrogen.
80 °C
25 °C
-80 °C
?. [3.2.2 Generation of RNA Transcript]
Incubate the reaction for – at .
37 33
?. [3.2.2 Generation of RNA Transcript]
Add  per 1 μg DNA.
[DNAse I]
?. [3.2.2 Generation of RNA Transcript]
Incubate for .
37 33
?. [3.2.3 RNA Isolation and Folding]
Overlay the pellet with  for  and incubate for  at .
[75% ethanol]
[TRIzol]
-20 °C
?. [3.2.3 RNA Isolation and Folding]
Centrifuge at . Discard the supernatant and air dry the pellet.
Centrifuge: 7500 34, 10 min, 4 10
?. [3.2.3 RNA Isolation and Folding]
Solve the pellet in water. Measure concentration using a NanoDrop and adjust the concentration to .
?. [3.2.3 RNA Isolation and Folding]
Incubate for  at .
0 Room temperature
?. [3.2.3 RNA Isolation and Folding]
Add  for  and mix carefully for  by inverting.
[chloroform]
[TRIzol ]
?. [3.2.3 RNA Isolation and Folding]
Incubate for  at .
0 Room temperature
?. [3.2.3 RNA Isolation and Folding]
Centrifuge at . Isolate the aqueous phase (see Note 7).
Centrifuge: 12000 34, 15 min, 4 10
?. [3.2.3 RNA Isolation and Folding]
Add  for  and mix carefully.
[isopropyl]
[TRIzol]
?. [3.2.3 RNA Isolation and Folding]
Incubate  at .
4 °C
?. [3.2.2 Generation of RNA Transcript]
Fill the reaction with water to the final volume of .
500 µl