Analysis of De Novo Synthesized Proteins
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 3.4 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 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
3.4.1 TCA Precipitation and Scintillation Counting
After the reaction is completed collect 2 × 3µL. Centrifuge the remaining mix at 16000x g,4°C and collect 2 × 3µL. Resuspend the microsomal fraction in an equal volume of PBS in comparison to the volume of the translation mixture. Collect 2 × 3µL.
Mix each aliquot with 3mL and incubate in a water bath at 80°C for 0h 15m 0s. Store the aliquots for 30°C On ice or 0h 15m 0s at 4°C.
The mixture is applied to a vacuum filtration system to separate non-incorporated 14C-leucine from the radioactively labeled protein. Filters with the collected protein are washed twice with TCA and twice with acetone. Dry the filters under the hood.
The filters are transferred into the scintillation vials and overlaid with 3mL. After an incubation time of 1h 0m 0s with gentle shaking, scintillation vessels are counted in scintillation counter.
3.4.2 In-Gel- Fluorescence and Autoradiography
For preparation of SDS-PAGE samples take a 5µL (part 3.3, section 3.3.1 "Fluorescent Labeling with Bodipy-TMRLysine", steps 3–5 ) or 10µL (part 3.3, section 3.3.3 "Site-Specific Incorporation of a Non-canonical Amino Acid with Subsequent Fluorescent Labeling and Microscopic Analysis", steps 13–17 ) of each prepared sample.
Add 45µL and 150µL to the 5µL or 10µL and incubate for 0h 15m 0s On ice. Keep the fluorescently labeled samples in dark during the whole procedure. Centrifuge the samples at 16000x g,4°C and discard the supernatant.
Dry the pellets for 1h 0m 0s at 45°C in a thermo mixer with a shaking speed of 1000rpm.
Resuspend the dried pellets in 20µL and load the samples on a prepared 10%. Use a ladder with fluorescently labeled bands. Run the gel.
Transfer the gel to the fluorescence imaging system and detect the labeled protein bands. For Bodipy-TMR-lysine use a 532 nm laser and a 580 nm emission filter. Sulfo-Cy5 can be detected with extinction at 633 nm and emission at 670 nm.
Afterwards dry the gel for 1h 0m 0s at 70°C using a unigeldryer. The dried gels are exposed on a phosphorscreen for minimal 3 days and read out using a multi-mode imager.
3.4.3 Confocal Laser Scanning Microscopy
For confocal laser scanning microscopy use 5µL in the microsomal fraction and dilute the sample in 20µL. Add the mixture to a μ-Ibidi-Slide.
Fix the slide. Use a plan-apochromat objective with a 60× or 100× magnification. Microsomal structures usually have a diameter of 1–10 μm.
Adjust the beam path to the coupled fluorescent dye. Standard dyes such as Cy5 and FITC usually have a preset configuration. Cy5 is excited at 633 nm and the emission is detected with a long-pass filter above a wavelength of 670 nm.
Adjust the microscope settings (laser intensity, gain master, focus, pinhole) according to the individual sample ( see Note 17 ).
