Sep 22, 2021

Public workspacePreparation of Suppressor tRNA

DOI
dx.doi.org/10.17504/protocols.io.bqnvmve6
  • Anne Zemella1,
  • Theresa Richter1,2,
  • Lena Thoring1,
  • Stefan Kubick1
  • 1Cell-free and Cell-based Bioproduction, Fraunhofer Institute for Cell Therapy and Immunology (IZI), Branch Bioanalytics and Bioprocesses (IZI-BB), Potsdam, Germany;
  • 2University of Potsdam, Potsdam, Germany
  • Springer Nature Books
Satyavati Kharde
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DOI: dx.doi.org/10.17504/protocols.io.bqnvmve6
External link: https://link.springer.com/protocol/10.1007/978-1-4939-9121-1_4
Protocol Citation: Anne Zemella, Theresa Richter, Lena Thoring, Stefan Kubick 2021. Preparation of Suppressor tRNA. protocols.io https://dx.doi.org/10.17504/protocols.io.bqnvmve6
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License,  which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
Created: December 12, 2020
Last Modified: September 22, 2021
Protocol Integer ID: 45493
Keywords: 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

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
Materials
2.2 Materials for Preparation of Suppressor tRNA

2.2.1 Generation of PCR Product

  1. Vector containing the nucleotide sequence of tRNATyrCUA (SupF Gene).
  2. tRNATyrCUA-specific forward primer (5’ CgA gCT CgC CCA CCg gAA TTC 3’) and 2’-OMe reverse primer (5’ Tgg Tgg Tgg ggg AAg gAT TCg 3’).
  3. 0.2 ml PCR tubes.
  4. PCR cycler.
  5. Taq DNA polymerase.
  6. Taq buffer.
  7. dNTPs.
  8. 25 mM MgCl2.
  9. Agarose gel electrophoresis chambers.
  10. Agarose.
  11. Rotiphorese 10× TBE buffer.
  12. DNA stain.
  13. DNA ladder.
  14. PCR Purification Kit.

2.2.2 Generation of RNA Transcript

  1. T7 RNA Polymerase (f.c. 1 U/μl, Agilent).
  2. 5× NTP mix containing 18.75 mM ATP, 18.75 mM CTP, 18.75 mM UTP and 7.5 mM GTP.
  3. 5× transcription buffer: 400 mM HEPES-KOH, 0.5 mM Spermidine, 50 mM DTE and 75 mM MgCl2.
  4. DNAseI (1 U per μg plasmid DNA).
  5. 10× MOPS buffer: 200 mM MOPS, 50 mM NaOAc, 10 mM EDTA, pH 8.0.
  6. MOPS sample buffer: 8% (v/v) formaldehyde, 12 ml formamide, 2.4 ml 10× MOPS buffer, 0.05% (v/v) bromophenol blue to a total volume of 24 ml.

2.2.3 RNA Isolation and Folding

  1. TRIzol reagent.
  2. High Performance Liquid Chromatography (HPLC) grade Chloroform.
  3. HPLC grade Isopropyl.
  4. 75% Ethanol.
  5. Cooled centrifuge.
  6. Nanodrop 2000c.
Safety warnings
For hazard information and safety warnings, please refer to the SDS (Safety Data Sheet).
3.2.1 Generation of PCR Product
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 Concentration1 x Taq Buffer , Concentration0.2 millimolar (mM) dNTP mix , Concentration0.5 micromolar (µM) forward primer , Concentration0.5 micromolar (µM) reverse primer , Concentration2.5 millimolar (mM) MgCl2 , Concentration0.01 ng/μl plasmid and Concentration0.04 U/μl Taq DNA polymerase . Fill the reaction with water to a final volume of Amount250 µL (see Note 5).
3.2.2 Generation of RNA Transcript
3.2.2 Generation of RNA Transcript
6h
6h
Thaw the components for in vitro transcription TemperatureOn ice and pipette the reaction at TemperatureRoom temperature . Mix Concentration1 x transcription buffer , Concentration1 x NTP mix , Concentration1 U/μl T7 RNA Polymerase and Concentration8 ng/μl template DNA
.
Pipetting
Fill the reaction with water to the final volume of Amount500 µL .
Mix
Incubate the reaction for Duration03:00:00 –Duration06:00:00 at Shaker500 rpm, 37°C .

6h
Incubation
Centrifuge the RNA at Centrifigation12000 x g, 00:01:00 und use the supernatant for the DNAseI treatment (see Note 6).
Centrifigation
Add Amount1 U DNAse I per 1 μg DNA.
Mix
Incubate for Shaker500 rpm, 37°C, 00:10:00 .

10m
Incubation
3.2.3 RNA Isolation and Folding
3.2.3 RNA Isolation and Folding
1h 41m 15s
1h 41m 15s

Safety information
Handle the TRIzol and chloroform reagent with care and use a fume hood.
Add a threefold volume of TRIzol to the transcription reaction and mix carefully.
Mix
Incubate for Duration00:05:00 at TemperatureRoom temperature .
5m
Incubation
Add Amount200 µL chloroform for Amount1 mL TRIzol and mix carefully for Duration00:00:15 by inverting.
15s
Mix
Incubate for Duration00:03:00 at TemperatureRoom temperature .
3m
Incubation
Centrifuge at Centrifigation12000 x g, 4°C, 00:15:00 . Isolate the aqueous phase (see Note 7).
Centrifigation
Add Amount500 µL isopropyl for Amount1 mL TRIzol and mix carefully.
Mix
Incubate DurationOvernight at Temperature4 °C .
3m
Incubation
Centrifuge at Centrifigation15000 x g at least for Duration01:00:00 at Temperature4 °C and discard the supernatant.
1h
Centrifigation
Overlay the pellet with Amount1 mL 75% ethanol for Amount1 mL TRIzol and incubate for Duration00:30:00 at Temperature-20 °C .
30m
Incubation
Centrifuge at Centrifigation7500 x g, 4°C, 00:10:00 . Discard the supernatant and air dry the pellet.
Centrifigation
Solve the pellet in water. Measure concentration using a NanoDrop and adjust the concentration to Concentration100 micromolar (µM) .
Imaging
Computational step
Fold the tRNA by slowly decreasing the temperature from Temperature80 °C to Temperature25 °C in a PCR cycler. The tRNA can be stored at Temperature-80 °C after shock freezing in liquid nitrogen.
PCR