TITLE: Preparation of Enhanced Orthogonal Aminoacyl-tRNA-Synthetase
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 3.1 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]
?. [3.1 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 ,  , , ,  for the induction of the protein expression pQE2 vector,   containing .
[E.coli lysate]
[reaction mix]
[amino acids without methionine]
[methionine]
[IPTG]
[template]
[plasmid DNA]
?. [3.1 Preparation of Enhanced Orthogonal Aminoacyl-tRNA-Synthetase]
The surrounding feeding mixture contains , ,  and  (see Note 2).
[feeding mix]
[IPTG]
[amino acids without methionine]
[methionine]
?. [3.1 Preparation of Enhanced Orthogonal Aminoacyl-tRNA-Synthetase]
Fill the reaction solution into the reaction compartment (marked through the red lid).
?. [3.1 Preparation of Enhanced Orthogonal Aminoacyl-tRNA-Synthetase]
Fill the feeding mix into the feeding chamber (marked through the colorless lid).
?. [3.1 Preparation of Enhanced Orthogonal Aminoacyl-tRNA-Synthetase]
Insert the prepared chamber into the RTS 500 adapter in a thermomixer. The reaction time is  at  and a shaking speed of .
30 °C
33
?. [3.1 Preparation of Enhanced Orthogonal Aminoacyl-tRNA-Synthetase]
For the separation of aggregated proteins from soluble eAzFRS a centrifugation step at  is recommended.
Centrifuge: 16000 34, 10 min, 4 10
?. [3.1 Preparation of Enhanced Orthogonal Aminoacyl-tRNA-Synthetase]
Equilibrate two Strep-Tactin columns with  and add  to each column.
[10× washing buffer]
[supernatant of the cell-free reaction]
?. [3.1 Preparation of Enhanced Orthogonal Aminoacyl-tRNA-Synthetase]
After the supernatant has completely entered the column, wash each column 5× with  (see Note 3).
[washing buffer]
?. [3.1 Preparation of Enhanced Orthogonal Aminoacyl-tRNA-Synthetase]
Elute the protein 6× with  and collect the fractions.
[elution buffer]
?. [3.1 Preparation of Enhanced Orthogonal Aminoacyl-tRNA-Synthetase]
Elution fractions containing the target protein are pooled.
?. [3.1 Preparation of Enhanced Orthogonal Aminoacyl-tRNA-Synthetase]
Regenerate the column with 3×  and remove the regeneration buffer 2× with . Store the column in  at .
[1× regeneration buffer]
[1× washing buffer]
[washing buffer]
4 °C
?. [3.1 Preparation of Enhanced Orthogonal Aminoacyl-tRNA-Synthetase]
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 . Add  to the resin bed and centrifuge at . Repeat this step 2×. Place the column in a new collection tube and apply  to each column. Centrifuge at  and collect the synthetase.
Centrifuge: 1500 34, 1 min
[synthetase storage buffer]
Centrifuge: 1500 34, 1 min
[pooled synthetase solution]
Centrifuge: 2000 34, 2 min
?. [3.1 Preparation of Enhanced Orthogonal Aminoacyl-tRNA-Synthetase]
The concentration of the synthetase can be performed with Amicon® Ultra Centrifugal Filters. Add up to  to the concentrator and centrifuge at . 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).
[synthetase solution]
Centrifuge: 14000 34, 10 min, 4 10
?. [3.1 Preparation of Enhanced Orthogonal Aminoacyl-tRNA-Synthetase]
The synthetase can be stored at  after shock freezing in liquid nitrogen.
-80 °C