Preparation of Enhanced Orthogonal Aminoacyl-tRNA-Synthetase
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 3.1 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 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
3.1 Preparation of Enhanced Orthogonal Aminoacyl-tRNA-Synthetase
For prokaryotic cell-free synthesis, the eAzFRS gene should be cloned into a vector containing a T7 promotor, ribosomal binding site, and T7 terminator such as pIX3.0, pIVEX2.3d, and pIVEX2.4d vectors or alternatively containing a T5 promotor such as pQE2 vectors as used in this protocol. eAzFRS is synthesized in a cell-free system using an E. coli lysate in a dialysis mode.
A typical 1.1 ml reaction is composed of 0.525mL, 0.225mL , 0.27mL, 30µL, 11µL for the induction of the protein expression pQE2 vector, 39µL containing 110µg.
The surrounding feeding mixture contains 7990µL, 110µL, 2650µL and 300µL ( see Note 2 ).
Fill the reaction solution into the reaction compartment (marked through the red lid).
Fill the feeding mix into the feeding chamber (marked through the colorless lid).
Insert the prepared chamber into the RTS 500 adapter in a thermomixer. The reaction time is 20h 0m 0s at 30°C and a shaking speed of 900rpm.
For the separation of aggregated proteins from soluble eAzFRS a centrifugation step at 16000x g,4°C is recommended.
Equilibrate two Strep-Tactin columns with 400µL and add 500µL to each column.
After the supernatant has completely entered the column, wash each column 5× with 200µL ( see Note 3 ).
Elute the protein 6× with 100µL and collect the fractions.
Elution fractions containing the target protein are pooled.
Regenerate the column with 3× 1mL and remove the regeneration buffer 2× with 800µL. Store the column in 2mL at 4°C.
The combined elution fractions are applied to Zeba™ Spin Desalting Columns to exchange the elution buffer of the strep-tag purification to a synthetase storage buffer. Therefore, remove the storage solution of the Zeba™ Spin Desalting Column by centrifugation at 1500x g. Add 300µL to the resin bed and centrifuge at 1500x g. Repeat this step 2× .
Place the column in a new collection tube and apply 100µL to each column. Centrifuge at 2000x g and collect the synthetase.
The concentration of the synthetase can be performed with Amicon® Ultra Centrifugal Filters. Add up to 500µL to the concentrator and centrifuge at 14000x g,4°C. Collect the concentrated sample and determine the concentration by NanoDrop measurement using the molecular mass (48.6 kDa) and the extinction coefficient (54.3) ( see Note 4 ).
The synthetase can be stored at -80°C after shock freezing in liquid nitrogen.
