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In This Article

  • Summary
  • Abstract
  • Introduction
  • Protocol
  • Results
  • Discussion
  • Disclosures
  • Acknowledgements
  • Materials
  • References
  • Reprints and Permissions

Summary

A facile fluorescence assay is presented to evaluate the efficiency of amino-acyl-tRNA-synthetase/tRNA pairs incorporating non-canonical amino-acids (ncAAs) into proteins expressed in mammalian cells. The application of ncAAs to study G-protein coupled receptors (GPCRs) is described, including photo-crosslinking mapping of binding sites and bioorthogonal GPCR labeling on live cells.

Abstract

The genetic incorporation of non-canonical amino acids (ncAAs) via amber stop codon suppression is a powerful technique to install artificial probes and reactive moieties onto proteins directly in the live cell. Each ncAA is incorporated by a dedicated orthogonal suppressor-tRNA/amino-acyl-tRNA-synthetase (AARS) pair that is imported into the host organism. The incorporation efficiency of different ncAAs can greatly differ, and be unsatisfactory in some cases. Orthogonal pairs can be improved by manipulating either the AARS or the tRNA. However, directed evolution of tRNA or AARS using large libraries and dead/alive selection methods are not feasible in mammalian cells. Here, a facile and robust fluorescence-based assay to evaluate the efficiency of orthogonal pairs in mammalian cells is presented. The assay allows screening tens to hundreds of AARS/tRNA variants with a moderate effort and within a reasonable time. Use of this assay to generate new tRNAs that significantly improve the efficiency of the pyrrolysine orthogonal system is described, along with the application of ncAAs to the study of G-protein coupled receptors (GPCRs), which are challenging objects for ncAA mutagenesis. First, by systematically incorporating a photo-crosslinking ncAA throughout the extracellular surface of a receptor, binding sites of different ligands on the intact receptor are mapped directly in the live cell. Second, by incorporating last-generation ncAAs into a GPCR, ultrafast catalyst-free receptor labeling with a fluorescent dye is demonstrated, which exploits bioorthogonal strain-promoted inverse Diels Alder cycloaddition (SPIEDAC) on the live cell. As ncAAs can be generally applied to any protein independently on its size, the method is of general interest for a number of applications. In addition, ncAA incorporation does not require any special equipment and is easily performed in standard biochemistry labs.

Introduction

The genetic incorporation of chemical probes into proteins is a powerful method to facilitate investigation of structural and dynamic aspects of protein function directly in the native context of the live cell. Nowadays, hundreds of non-canonical amino acids (ncAAs) equipped with the most disparate chemical groups can be site-specifically incorporated into proteins by biosynthesis1,2,3,4. Between them, one finds photo-sensitive ncAAs such as photo-crosslinkers5, photo-caged6,7,8,9 and photo-switchable amino acids10,11, amino acids bearing strained alkenes and alkynes for catalyst-free bioorthogonal chemistry2,12,13,14,15,16,17, amino acids carrying dansyl18, coumarin9,19, and prodan20,21 fluorophores, and amino acids equipped with other biophysical probes as well as with post translational modifications1,2,3,4,22,23,24,25.

The genetic encoding of a ncAA is enabled by a dedicated amino-acyl-tRNA-synthetase (AARS) paired to a cognate suppressor-tRNA, which incorporates the ncAA in response to an amber stop codon during the regular ribosomal synthesis. ncAARS/tRNA pairs are engineered so as to be orthogonal in the host organism, i.e. not cross-talk with the endogenous pairs. The technique is well established both in prokaryotic and eukaryotic hosts and easily applicable to mammalian cells. Pairs for ncAA incorporation in mammalian cells are based on three main orthogonal systems: the tyrosyl system, that combines the TyrRS from E. coli26 with a tyrosyl amber suppressor from B. stearothermophilus27 (EcTyrRS/BstYam pair), the E. coli leucyl system (EcLeuRS/tRNALeuCUA pair)6,18,28 and the archaeal pyrrolysyl system (PylRS/tRNAPyl pair)3, whereby the tRNAPyl is a natural amber suppressor. In general, each ncAA is recognized by a specialized ncAARS. Depending on the structure of the ncAA, the ncAARS is obtained via directed evolution of either TyrRS, LeuRS or PylRS, although some synthetases can accept more than one ncAA.

The orthogonal pair is imported into the cells by simply using a plasmid vector. Most common and efficient plasmids are bicistronic and encode both for the synthetase and the tRNA forming the orthogonal pair29. A second plasmid encoding for the protein of interest bearing an amber codon at the site designated for modification is co-transfected. The ncAA is simply added to the cell growth medium. However, different specialized groups often use different variants of plasmid constructs even for the incorporation of the same ncAA. Constructs differ in the arrangement of the genes in the vector, type of the synthetase, codon usage in the synthetase gene, promoter usage, variant of the tRNA and number of tRNA expression cassettes. Moreover, the incorporation efficiency of different ncAAs can vary drastically due to the different catalytic efficiency of the different synthetases, the quality of the tRNA, and other factors30. Therefore, it is important to have at hand a fast and reliable method to evaluate the efficiency of an orthogonal pair, both to choose the most suitable system for a desired application and to perform some optimization steps that improve overall protein expression yields.

We have established a simple and robust fluorescence-based assay to evaluate the efficiency of orthogonal pairs29 (Figure 1). In the assay, cells are co-transfected with the plasmid encoding for the orthogonal pair, together with a bicistronic reporter plasmid encoding both for the green fluorescent protein bearing an amber stop codon at a permissive position (EGFPTAG) and the mCherry gene. Red and green fluorescence of whole-cell lysates are read in separate channels on a plate reader in a 96-well plate. The intensity of the green fluorescence directly correlates with the efficiency of amber suppression, whereas the intensity of red fluorescence gives a direct estimate of the size of the measured sample and the transfection efficiency. With respect to similar assays based on fluorescence assisted cell sorting (FACS) read out31,32, the assay gives an immediate and comprehensive assessment of protein expression in the whole cell population, which is more representative of usual experimental conditions, and offers an easier data acquisition and processing with standard software. Overall, the main advantage of the assay is that a medium to a large number of samples can be analyzed in parallel. Using this assay, we have screened a rationally designed library of suppressor-tRNAs to improve the efficiency of the Pyl orthogonal system30. This work describes the experimental protocol to perform this assay and show examples of its application, including the optimization of the orthogonal pair for the incorporation of the photo-crosslinking ncAA p-azido-L-phenylalanine (Azi) and the comparison of incorporation efficiencies of different amino acids (Figure 2).

Over the last years, ncAA tools have been proven very powerful to investigate structural and functional aspects of G-protein coupled receptors (GPCRs)33,34,35,36,37,38. In humans, GPCRs form a large family of membrane receptors (800 members) and represent main targets for therapeutic drugs. Direct structural characterization of GPCRs is still challenging and complementary biochemical methods are highly needed for their investigation. We have pioneered the use of photo-crosslinking ncAAs to map GPCR surfaces and discover ligand binding pockets34. Using our optimized system for Azi incorporation, we systematically incorporated Azi throughout the whole juxtamembrane domain of a GPCR directly in live mammalian cells. Upon UV irradiation, Azi forms a highly reactive nitrene species that covalently captures neighboring molecules. When the ligand is added to the system, Azi serves as a proximity probe to reveal which positions of the receptor come close to the bound ligand. In this way, the binding mode of the neuropeptide hormone Urocortin I (Ucn1) on the class B GPCR corticotropin-releasing-factor receptor type 1 (CRF1R)33 was first unveiled. Lately, we have disclosed distinct binding patterns of agonists and antagonists on the same receptor38. A similar approach has been applied by others to reveal orthosteric and allosteric binding sites of other peptides and small molecule ligands on other GPCRs39,40,41,42. This manuscript describes the experimental protocol applied in our lab for photo-crosslinking mapping of GPCR surfaces. The method is relatively fast, straightforward and does not require any special equipment, so that it is applicable in standard biochemistry labs. Importantly, the approach provides a valuable tool not only to identify ligand binding sites where 3D structural data are scarce, but also to supplement existing in vitro data with information from fully post-translationally modified receptors in the physiological environment of the live cell.

The recent development of novel ncAAs bearing on the side chain chemical groups suitable for ultrafast catalyst-free bioorthogonal chemistry has opened up the possibility to install last-generation fluorophores for super-resolution imaging into proteins directly on the live cells2,43. Such chemical anchors include strained cyclooctyne in SCOK14, bicyclo[6.1.0]nonyne in BCNK12,17, and trans-cyclooctenes in TCO*K13,15,17 among other ncAAs harboring a norbornene16,17,44 or cyclopropene45,46 moiety. Bulky ncAAs for bioorthogonal chemistry are incorporated by a variant of the PylRS usually denoted as PylRSAF (indicating mutation Y271A and Y349F in M. barkeri PylRS), as well as by other ad hoc evolved ncAARSs17,44. The bioorthogonal anchors react with tetrazine reagents47 via inverse electron-demand Diels-Alder cycloaddition to give high labeling yields within a few minutes43,48. However, application of this powerful approach to label GPCRs has been challenging due to a low overall efficiency of the orthogonal ncAA incorporation system. Using our enhanced Pyl system, we have recently demonstrated high-yield incorporation of such amino acids into GPCRs and ultrafast GPCR labeling on the surface of live mammalian cells30. Labeled receptors were still functional, as they physiologically internalized upon activating the receptor with an agonist. The experimental protocol for the incorporation of bioorthogonal anchors into GPCRs and the following labeling steps are described here. Equipping GPCRs with small bright fluorophores is the first fundamental step toward the study of GPCR structural dynamics in the live cell via advanced microscopy techniques.

Protocol

1. Fluorescence-based Screening of Incorporation Efficiencies (Figure 1)

  1. Maintain HEK293 cells in Dulbecco's Modified Eagle's Medium (DMEM; high glucose, 4 mM glutamine, pyruvate) supplemented with 10 % (v/v) fetal bovine serum (FBS), 100 U/mL penicillin and 100 µg/mL streptomycin at 37 °C, 95 % humidity and 5 % CO2.
  2. Seed the cells the day before transfection.
    1. Detach the cells for 5 min at 37 °C in 0.05 % Trypsin/PBS supplemented with 0.5 mM EDTA. Use 1 mL Trypsin/EDTA for a 10-cm dish. Quench with 10 volumes of complete medium and resuspend the cells by pipetting. Count the number of cells in the suspension using a hemocytometer49.
    2. Seed 6.0 x 105 HEK293 cells per well of 6-well plates in 2 mL complete growth medium. Prepare as many wells as the number of samples, and two additional wells for the wild-type EGFP and a mock-transfected sample, respectively.
  3. Control confluence (area occupied by the cells) under a microscope. Transfect cells at ~70 % confluence using polyethyleneimine (PEI) reagent.
    1. 1h prior to transfection, add the appropriate amount of freshly prepared ncAA stock solution to all wells for a final ncAA concentration of 0.25-0.5 mM. Add the ncAA to all wells, including the wild-type positive control and mock-transfected cells, to prevent differences in fluorescence signals that may be caused by effects of the ncAA on cellular growth.
      Note: To prepare stock solutions, dissolve the ncAA to 0.1-0.5 M using 0.2-0.5 M NaOH. However, some ncAAs may require initial solubilization in DMSO and/or neutralization by four volumes of 1 M HEPES (pH 7.4) before use. Commonly, the manufacturer recommends a protocol to prepare a stock solution.
    2. In a microcentrifuge tube, mix 1 µg of plasmid DNA encoding for the ncAARS/tRNA pair to be tested with 1 µg of reporter plasmid DNA (pcDNA3.0-EGFP183TAG-mCherry). In separate tubes, prepare an identical transfection using the EGFP wild-type reference and a mock transfection.
      Note: Number of copies of the tRNA cassette embedded in the plasmid encoding for the ncAARS/tRNA pair depends on the application. To facilitate cloning, 1 tRNA copy is recommended when screening different tRNAs, whereas 4 copies are recommended (albeit not strictly necessary) when either testing different ncAARS or the incorporation of different ncAAs by the same orthogonal pair.
    3. To each tube containing the DNA add 100 µL lactate buffered saline (LBS) containing 20 mM sodium lactate at pH 4.0 and 150 mM NaCl. Mix briefly.
    4. To each tube containing the DNA in LBS add 6 µL of 1 µg/µL PEI in LBS (ratio PEI/DNA = 3/1 w/w) and vortex immediately. Incubate at RT for 10-15 min.
    5. Take 400 µL cell medium from each well and add it to the DNA-PEI mixture to neutralize the pH. Dribble the DNA mixture onto the cells.
      Note: DMEM usually contains phenol red as pH indicator. During the neutralization step the color of the mixture added in the tube will change from yellow (acidic) to red (neutral). Although forming the DNA complexes in LBS at acidic pH gives the highest transfection yields50, DNA-PEI complexes can alternatively be formed directly at pH 7.4 (for instance in serum-free DMEM). If using DMEM to form DNA complexes, skip the neutralization step 1.3.5. In any case, it is essential that no serum is present in the mixture when forming the complexes.
  4. Harvest cells 48 h post-transfection.
    1. Aspirate the medium and rinse the cells once with 2 mL pre-warmed PBS (37 °C). Add 800 µL of PBS supplemented with 0.5 mM EDTA and incubate for 20 min at 37 °C. Detach and suspend the cells by pipetting up and down.
    2. Transfer the cell suspension into 1.5 mL tubes containing 200 µL PBS supplemented with 5 mM MgCl2.
    3. Centrifuge for 2 min at 800 x g and discard the supernatant.
      Note: The protocol can be paused here. In this case, flash-freeze the pellets in liquid N2 and store at -80 °C for up to one month. Always wear eye protection goggles.
  5. Add 100 µL Tris lysis buffer (50 mM Tris-HCl pH 8.0, 150 mM NaCl, 1% Triton X-100, 1 mM EDTA and freshly added PMSF) to the cell pellets and incubate on ice for 30 min. To facilitate lysis, vortex every 5 min.
  6. Spin down the cell debris for 10 min at 4 °C and 14,000 x g and transfer 90 µL of the supernatant into black 96-well plates. Measure EGFP and mCherry fluorescence using a plate reader equipped with a fluorescence module.
    Note: Use appropriate excitation and emission filters for EGFP (λabs: 488 nm; λem: 509 nm) and mCherry (λabs: 588 nm; λem: 611 nm). Measured EGFP values will span a range between the minimum value obtained from mock-transfected cells and a maximum value, which is usually obtained from wild-type EGFP. Take care of setting up the correct measurement window in the instrument.
  7. The efficiency of ncAA incorporation is calculated as the ratio between the fluorescence of the sample and the fluorescence obtained from expression of wild-type EGFP. All values are normalized to mCherry fluorescence.
    figure-protocol-5605

2. Genetic Incorporation of ncAAs into GPCRs for Photo-crosslinking Mapping of Ligand-GPCR Interactions (Figure 3)

  1. Maintain HEK293T cells in DMEM supplemented with 10 % (v/v) FBS, 100 U/mL penicillin and 100 µg/mL streptomycin at 37 °C, 95 % humidity and 5 % CO2.
  2. Seed cells the day before transfection.
    1. Detach the cells for 5 min at 37 °C in 0.05 % Trypsin/PBS supplemented with 0.5 mM EDTA. Use 1 mL Trypsin/EDTA for a 10-cm dish. Quench with 10 volumes of complete medium and resuspend the cells by pipetting up and down. Count the number of cells in the suspension using a hemocytometer49.
    2. Seed 5.0 x 105 293T cells per well in 2 mL complete growth medium in 6-well plates. For each position to be screened, prepare 1 well per ligand plus one well for the binding control33,38. An extra well to be transfected with the wild-type (wt) receptor may be included to check the expression level of the mutant.
  3. The day after, control confluence (area occupied by the cells) under a microscope. Transfect cells at ~70% confluence using PEI.
    1. 1h prior to transfection, add Azi to all wells to a final concentration of 0.5 mM.
      1. Prepare a 0.5 M stock solution of Azi. Per 6-well plate, weigh 1.2 mg Azi into a tube and dissolve it in 15 µL 0.5 M NaOH. Dilute the stock solution in 1.2 mL complete medium and add 200 µL of the mixture to each well.
        Note: Prepare a fresh stock solution of Azi for every experiment. The azide moiety has a short half-life in aqueous solutions, especially at basic pH, and the AziRS incorporates the intact but also the degraded form.
    2. Transfect a total amount of 2 µg DNA per well: 1 µg of plasmid encoding for the FLAG-tagged GPCR bearing a TAG codon at the desired position and 1 µg of the plasmid encoding for the orthogonal pair dedicated to Azi (E2AziRS51 and 4 copies of the cognate suppressor-tRNA BstYam)33,38.
      Note: When including a wt comparison to check expression levels, transfect a lower amount of plasmid DNA for the wt receptor. Depending on the GPCR, 0.2-0.5 µg of plasmid encoding the wt receptor yield similar levels as 1.0 µg of the mutant plasmid. Transfect the same amount of DNA in all wells, filling up the missing DNA with a mock (for instance an empty vector).
    3. Proceed as described in 1.3.3-1.3.5.
    4. 48 h post-transfection, proceed either with step 2.4 for photo-crosslinking of the ligands or go to step 2.5 for direct harvesting and analysis for verifying receptor expression.
  4. Photo-crosslinking of the ligand.
    1. Prepare a 1,000x ligand stock solution. Dissolve the peptide ligand at a concentration of 100 µM in DMSO.
      Note: The ligand concentration depends on the dissociation constant KD of the ligand-GPCR interaction. A final concentration of 100 x KD is recommendable. If the peptide ligand is a salt of trifluoroacetic acid (TFA), consider the weight of TFA when calculating the molecular weight (1 x TFA per basic amino acid in the peptide). Also, consider that peptides are in general hygroscopic. Avoid repeated freezing of peptide powder and never open a peptide container until it has not reached room temperature.
    2. Dilute the ligand stock solution 1:1,000 in binding buffer consisting of 0.1% BSA, 0.01% Triton-X 100, 5 mM MgCl2 in HEPES dissociation buffer (HDB) containing 12.5 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES)-HCl pH 7.4, 140 mM NaCl and 5 mM KCl. Prepare 1 mL per Azi-GPCR mutant. Replace the cell medium with 1 mL of the ligand solution. Incubate for 10 min at RT.
      Note: Adjust the incubation time to the specific GPCR, accounting for ligand kinetics and receptor internalization. Prolonging the incubation time does not improve crosslinking yields.
    3. Irradiate the samples for 20 min in a UV crosslinker at 365 nm with 5 x 8 W tubes and ~ 5 cm distance to the cells. Detach the cells by pipetting and transfer them into a 1.5 mL reaction tube. Pellet the cells for 3 min at 800 x g and discard the supernatant.
    4. Dissolve a tablet of protease inhibitor (PI) cocktail in 1 mL 25 mM EDTA/H2O to make a 50x stock solution. Aliquot the PI stock solution and store it at -20 °C. Dilute the 50x stock 1:25 in HDB and resuspend the cell pellets in 50 µL of 2 x PI in HDB. Flash-freeze the cells in liquid N2.
      Note: Wear eye protection goggles. At this point, the samples can be stored at -80 °C for up to one month. Proceed with step 2.6.
  5. Direct cell harvest.
    1. Aspirate the medium. Add 800 µL of 0.5 mM EDTA in HDB. Incubate for 10 min at RT or on ice.
    2. Detach the cells by pipetting up and down and transfer them into a 1.5 mL reaction tube. Add 200 µL of 5 mM MgCl2 in HDB. Pellet the cells for 3 min at 800 x g and discard the supernatant.
    3. Resuspend the cell pellets in 50 µL of 2 x PI in HDB and flash freeze in liquid N2. Wear eye protection goggles.
      Note: At this point, the samples can be stored at -80 °C for up to 1 month.
  6. Cell lysis.
    1. Thaw the cells in a water bath at 37 °C for 30-45 s and vortex briefly. Keep samples cold from now on. Pellet membranes at 2,500 x g and 4 °C for 10 min. Discard the supernatant, which contains the bulk of cytosolic proteins.
    2. Resuspend the pellets in 50 µL HEPES lysis buffer containing 50 mM HEPES-HCl pH 7.5, 150 mM NaCl, 10 % glycerol, 1 % Triton X-100, 1.5 mM MgCl2, 1 mM EGTA, 1 mM DTT and freshly added 2 x PI cocktail. Mix thoroughly. Lyse the cells 30 min on ice and vortex every 5 min.
    3. Spin down the cell debris for 10 min at 14,000 x g and 4 °C. Immediately transfer the supernatant to a fresh reaction tube.
      Note: Proceed with the analysis right away. The lysates can be stored at -20 °C, however, every freeze-thaw cycle impairs the quality of the results.
  7. Western blot analysis.
    1. To prepare the sample, take 3-5 µL lysate and fill it up to 7 µL with H2O. Add 2 µL 1 M DTT and 3 µL 4 x sample buffer containing 63 mM Tris-HCl pH 6.8, 2 % SDS, 10 % glycerol and 0.04 % bromphenol blue. Incubate for 30 min at 37 °C.
    2. When the GPCR is glycosylated and faint or smeared bands are a problem, deglycosylate samples with PNGase F to increase signal intensity and sharpen the bands. Use 3-5 µL lysate and deglycosylate in a total volume of 10 µL following the supplier's protocol. Add 3 µL 4 x sample buffer.
      Note: Membrane proteins are often glycosylated in multiple sites and states, which impairs the quality of resolution in SDS-PAGE analysis. However, do not deglycosylate the samples for analysis of the expression level of the Azi-GPCR mutants using anti-FLAG antibodies because it is relevant to evaluate the portion of the fully glycosylated, mature receptor at the cell surface.
    3. Resolve samples via standard SDS-PAGE and blot transfer proteins to a PVDF membrane.
      CAUTION: Acrylamide is neurotoxic. Wear gloves and eye protection.
    4. Block the membrane for 1 h at RT or overnight at 4 °C in 5 % skim milk in TBS-T containing 20 mM Tris-HCl pH 7.4, 0.15 M NaCl and 0.1 % Tween 20.
    5. Probe the membrane with an anti-ligand antibody followed by the HRP-conjugated secondary antibody. Wash in between with TBS-T. To detect the expression level of the Azi-GPCR, probe the membrane with a commercial HRP antibody (see Table of Materials).
    6. Perform enhanced chemiluminescence (ECL) reaction using homemade ECL reagent and detect signals for 5 min in the dark.

3. Ultrafast Bioorthogonal Labeling of GPCRs on Live Mammalian Cells

Note: The protocol is optimized for 4-well chambered coverslips (well area = 2.2 cm2). For different well sizes, the protocol must be scaled accordingly.

  1. Surface coating of microscope slides. Carry out the whole procedure under a sterile hood.
    1. Prepare a poly-D-lysine hydrobromide (MW=500-550 kDa) (PDL) stock solution at a concentration of 1 mg/mL in 50 mM borate buffer (pH 8.5). Store at 4 °C for up to 6 months. Do not freeze.
    2. Dilute the PDL stock solution 1:40 in sterile ultra-pure water to a final concentration of 25 µg/mL (working solution), then filter the solution through a 0.22 µm sterile filter.
      Note: The working solution can be stored at 4 °C for up to 3 months.
    3. Fully cover the bottom of each well of the microscopy slide with 500 µL of PDL working solution. Incubate for 20 min at RT and aspirate the PDL working solution.
      Note: The PDL working solution can be used up to three times. If the solution needs to be reused, transfer the used solution from the coated slides to a fresh sterile tube and label the tube accordingly. Never mix the recycled solution with fresh solution.
    4. Rinse each well 3 x with ~ 700 µl sterile ultra-pure water and let dry for at least 1 h.
      Note: It is very important to rinse the wells accurately, as residues of the PDL solution are toxic to the cells. The coated slides can be used straight away for microscopy or stored for up to one week at 4 °C.
  2. Maintain HEK293T cells in DMEM supplemented with 10 % (v/v) FBS, 100 U/mL penicillin and 100 µg/mL streptomycin at 37 °C, 95 % humidity and 5 % CO2.
  3. Seed cells the day before transfection.
    1. Detach the cells for 5 min at 37 °C in 0.05 % Trypsin/PBS supplemented with 0.5 mM EDTA. Use 1 mL Trypsin/EDTA for a 10-cm dish. Quench with 10 volumes of complete medium and resuspend the cells by pipetting. Count the number of cells in the suspension using a hemocytometer49.
    2. Seed 1.0 x 105 HEK293T cells per well (area 2.2 cm²) in 600 µL dye free complete DMEM.
      Note: For imaging purposes, it is very convenient to work from the beginning in a medium that does not contain any dye. Dye free DMEM formulations are commercially available.
  4. Control confluence (area occupied by the cells) under a microscope and transfect the cells at ~70 % confluence using a lipid-based transfection reagent.
    1. 1 h prior to transfection, prepare a fresh 100 mM stock solution of TCO*K in 0.2 M NaOH and 15 % (v/v) DMSO.
    2. Per well, mix 3 µl of the TCO*K stock solution with 12 µL of 1 M HEPES pH 7.4. Gently add the solution to the wells for a final TCO*K concentration of 0.5 mM.
    3. Prepare a total amount of 500 ng DNA per well. In a microcentrifuge tube, dilute 200 ng of pcDNA3.1_CRF1R-95TAG-EGFP, 200 ng of the plasmid encoding for the MbPylRSAF/tRNAPyl orthogonal pair (four cassettes of tRNAM15) and 100 ng of pcDNA3.1_Arrestin3 plasmid in 50 µL medium (dye free, serum-free, antibiotic free).
      Note: In general, co-transfection of Arrestin is not necessary to observe GPCR internalization. However, co-transfecting Arrestin3 speeds up internalization of CRF1R, which is very convenient when analyzing internalization of many mutants.
    4. Dilute 1.25 µL of the lipid-based transfection reagent (2.5 µL per 1 µg of DNA) in 50 µL medium (dye free, serum-free, antibiotic free) and add the solution to the DNA mixture. Vortex immediately and incubate 5-10 min at RT. Add DNA-lipid complexes to the cells.
      Note: In our experience, the morphology of cells transfected using lipid-based transfection looks more physiologic compared to that of cells transfected with PEI. As PEI gives higher transfection efficiency, PEI should be preferred for downstream applications like Western blot, whereas lipid-based transfection is a better choice to transfect cells for imaging experiments.
  5. 24 h post-transfection, label the receptor with fluorescent dyes.
    1. Prepare a 0.5 mM dye-tetrazine stock solution in DMSO and a 10 mg/mL of DNA staining dye stock solution in ultra-pure H2O.
    2. Transfer 100 µL medium from each well into a 1.5 mL reaction tube. Add 1.8 µL of the dye-tetrazine stock solution and 0.3 µL of the DNA staining dye stock solution. Transfer the medium containing the dyes back to the well and incubate for 5 min at 37 °C.
      Note: Tetrazine-orange-fluorescent dye has a final concentration of 1.5 µM.
    3. Aspirate the medium and gently rinse the cells twice with PBS to remove excess of dye. Add 600 µL of complete dye free growth medium preheated to 37 °C.
  6. Fluorescence microscopy and receptor internalization.
    1. Visualize the labeled receptors under 63x (or similar) magnification using filters appropriate for GFP (λabs: 488 nm; λem: 509 nm), orange-fluorescent dye (λabs: 550 nm; λem: 570 nm) and DNA staining dye (λabs: 350 nm; λem: 461 nm). Take a picture with each filter before activating the receptor.
    2. Promote receptor internalization using 200 nM of Ucn1.
      1. Prepare a 1,000 x Ucn1 stock solution of 200 µM in DMSO.
        Note: Depending on the solubility of the peptide, you may be able to prepare the stock in pure water or buffer.
      2. Transfer 100 µL medium from a well into a 1.5 mL reaction tube and add 0.6 µL of the peptide agonist stock solution. Transfer the medium back into the well.
      3. Observe the internalization under the microscope. Take pictures after the clearly detectable occurrence of internalization (10-15 min to hours, depending on the receptor and overexpression of Arrestins) using the filters mentioned previously.

Results

The outline of the fluorescence assay is depicted in Figure 1. The assay is employed in three applications. In first place, a number of tRNA variants for incorporation of Lys(Boc) by the Pyl orthogonal pair are screened. Lys(Boc) is an amino acid sterically similar to Pyl. As Pyl is not commercially available, Lys(Boc) is commonly used as a standard substrate for the PylRS. The screened tRNAs are based on the tRNAPyl. Each tRNA variant bears mutati...

Discussion

The protocol describes a simple and reliable assay to assess the efficiency of orthogonal pairs for the incorporation of ncAAs into proteins expressed in mammalian cells. The main advantage of this method in respect to widely used assays based on FACS is that it allows the simultaneous preparation and measurement of larger numbers of samples, and provides data that are easily analyzed using an ordinary software. The availability of a medium-throughput method to analyze orthogonal pairs in mammalian cells is very importan...

Disclosures

The authors have no conflicts to declare.

Acknowledgements

This work has been founded by the Deutsche Forschungsgemeinschaft (DFG) under grants CO822/2-1 (Emmy-Noether program) and CO822/3-1 to I.C.

Materials

NameCompanyCatalog NumberComments
Chemicals
Acryamide/Bisacrylamide 30% (37,5:1)Carl Roth3029.1
Ammonium persulfate (APS)Carl Roth9592.2
p-Azidophenylalanine (Azi)BachemF-3075.0001
Boric acidSigma AldrichB6768
BromphenolblueSigma-AldrichB0126-25G
Bovine serum albumine (BSA)Carl Roth8076.2
Carbobenzyloxy-L-lysine (Lys(Z))NovaBiochem8540430100
Cyclooctyne-L-lysine (SCOK)SichemSC-8000
DMEMLife Technologies41966052
DMSOCarl RothA994.2
DTTCarl Roth6908.1
enhanced chemiluminescence reagent (ECL) home-made10 mg/l luminol in 0.1 M Tris-HCl pH 8.6 ; 1100 mg/l  p-coumaric acid in DMSO ; 30 % H2O2 (1,000 : 100 : 0.3)
EDTACarl Roth8043.1
EGTACarl Roth3054.1
endo-bicyclo[6.1.0]nonyne-L-lysine (BCNK)SichemSC-8014
FBSThermo Fisher (Gibco)10270106
FluoroBrite DMEMThermo Fisher (Gibco)A1896701
GlycerolCarl Roth7533.1
GlycinCarl Roth3908.3
HEPESCarl Roth9105.3
Hoechst 33342Sigma AldrichB2261
KClCarl Roth6781.3
Lipofectamine 2000 Thermo Fisher11668019
LuminolApplichemA2185,0005
MethanolCarl Roth0082.3
MgCl2Carl Roth2189.2
NaClCarl RothHN00.2
Na-LactateSigma-Aldrich71718-10G
NaOHGrüssing121551000
PBSSigma-AldrichP5493-1L
p-Coumaric acidSigma-AldrichC9008-1G
poly-D-lysine hydrobromideCorning354210
PEIPolysciences23966
Penicillin/StreptomycinThermo Fisher (Gibco)11548876 (15140-122)
PMSFCarl Roth6367.1
PNGase FNEBP0704L
Protease InhibitorRoche11873580001
PVDF membrane Immobilon-PMilliporeIPVH00010
Skim Milk Powder Sigma70166
Sodium dodecyl sulfate (SDS)Carl RothCN30.2
Tetrazine-Cy3Jena BioscienceCLK-014-05
Tetramethylethylenediamine (TEMED)Carl Roth2367.3
trans-Cyclooctene-L-lysine (TCO*K)SichemSC-8008
TRISSigma-AldrichT1503
Triton X-100Carl Roth3051.4
Trypsin 2.5%Thermo Fisher (Gibco)15090046
Tween 20Carl Roth9127.2
Wasserstoffperoxid (30%)Merck1.07210.0250
Cell lines
HEK293 cellsGerman Collection of Microorganisms and Cell Cultures GmbH (DSMZ)ACC-305
HEK293T cellsGerman Collection of Microorganisms and Cell Cultures GmbH (DSMZ)ACC-635
Equipment
Crosslinker Bio-Link 365 nmBio-Budget Technologies GmbH40-BLX-E3655 x 8 Watt tubes
Plate Reader BMG LABTECH FLUOstar Omega BMG LABTECH
Plasmids
Plasmid E2AziRSThe huminized gene for E2AziRS was synthesized by Geneart (Life Technologies)Plasmid containing 4 tandem copies of the suppressor tRNA Bst-Yam driven by the human U6 promoter and one copy of a humanized gene for the enhanced variant of the Azi-tRNA synthetase (EAziRS) driven by a PGK promoter
POI-TAG mutant plasmidsPlasmid encoding the POI driven by the CMV promoter, C-terminally fused to the FLAG-tag, bearing a TAG codon at the desired position 
CRF1R-95TAG-EGFPCloned in the MCS of pcDNA3.1
HA-PTH1R-79TAG-CFPCloned in the MCS of pcDNA3.1
Arrestin3-FLAGSynthesized by Genart (Life Technologies)Cloned in the MCS of pcDNA3.1
Antibodies
Anti-FLAG-HRP M2 antibody conjugate Sigma-AldrichA8592 monoclonal, produced in mouse clone M2
Goat-anti-rabbit-HRP antibodySanta Cruzsc-2004
Rabbit-anti-CRF antibodyhome-madePBL #rC69polyclonal
Turnbull, A.V., Vaughan, J., Rivier, J.E., and Vale, W.W. Endocrinology, 140, (1), 71-78 (1999)
Rabbit-anti-Ucn1 antibodyhome-madePBL #5779polyclonal
Turnbull, A.V., Vaughan, J., Rivier, J.E., and Vale, W.W. Endocrinology, 140, (1), 71-78 (1999)

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