ABSTRACT
Genetic code expansion via stop codon suppression is a powerful technique for engineering proteins in mammalian cells with site-specifically encoded non-canonical amino acids (ncAAs). Current methods rely on very few available tRNA/aminoacyl-tRNA synthetase pairs orthogonal in mammalian cells, the pyrrolysyl tRNA/aminoacyl-tRNA synthetase pair from Methanosarcina mazei (Mma PylRS/PylT) being the most active and versatile to date. We found a previously uncharacterized pyrrolysyl tRNA/aminoacyl-tRNA synthetase pair from the human gut archaeon Methanomethylophilus alvus Mx1201 (Mx1201 PylRS/PylT) to be active and orthogonal in mammalian cells. We show that the new PylRS enzyme can be engineered to expand its ncAA substrate spectrum. We find that due to the large evolutionary distance of the two pairs, Mx1201 PylRS/PylT is partially orthogonal to Mma PylRS/PylT. Through rational mutation of Mx1201 PylT, we abolish its non-cognate interaction with Mma PylRS, creating two mutually orthogonal PylRS/PylT pairs. Combined in the same cell, we show that the two pairs can site-selectively introduce two different ncAAs in response to two distinct stop codons. Our work expands the repertoire of mutually orthogonal tools for genetic code expansion in mammalian cells and provides the basis for advanced in vivo protein engineering applications for cell biology and protein production.
INTRODUCTION
Genetic code expansion allows for the addition of new chemical functionalities to proteins in living cells in the form of non-canonical amino acids (ncAAs). ncAAs are site-specifically installed through repurposing of a genetically encoded nonsense stop codon, most often amber (TAG). So-called amber suppression relies on introduction of a tRNACUA/aminoacyl-tRNA synthetase pair into the cell that is orthogonal to, i.e. does not cross-react with, all endogenous tRNAs and aminoacyl-tRNA synthetases.
Nature created a repertoire of alternatives to the standard genetic code over billions of years of evolution. It is the rare outliers to the universal code that have provided useful molecular tools for synthetic biology (Baranov et al. 2015). The pyrrolysyl-tRNA (PylT)/pyrrolysyl-tRNA synthetase (PylRS) pair has become the most versatile tool for genetic code expansion in E. coli, yeast, mammalian cells and metazoan organisms. Pyrrolysine (Pyl, N-ε-4-methyl-pyrroline-5-carboxylate-L-lysine) is a biosynthetic amino acid, genetically encoded in a small number of methanogenic bacteria and archaea. In these organisms, a dedicated PylRS/PylTCUA pair directs Pyl incorporation in response to amber stop codons (Polycarpo et al. 2004). PylRS, the PylS gene product, accepts a range of structurally similar ncAAs in addition to its natural substrate. Further, PylRS has been a successful target for rational protein design and directed evolution, expanding the repertoire of accepted ncAA substrates (Wan et al. 2014). This includes ncAAs that carry chemical groups for bioorthogonal reactivity; photocaged amino acids for or photoactivated crosslinkers for photocontroled reactions; amino acids with native post-translational modifications (PTMs) (Chin 2017). The PylRS/PylT pair supports highly efficient recoding in mammalian cells (Elsässer et al. 2016;Schmied et al. 2014) enabling application of genetic code expansion technology to address biological questions in the context of the living cell.
In principle, reassignment of more than one natural codon could further augment the ability to engineer proteins harboring multiple ncAAs in vivo. Since PylT is not hardwired to recode amber codons, other stop codons can be recoded in mammalian cells (Ambrogelly et al. 2007;Xiao et al. 2013). Limiting to this application is the availability of mutually orthogonal tRNA/aminoacyl-tRNA synthetase pairs that are also orthogonal to the host cell. Thus, a key aim in the field is to find or rationally generate new mutually orthogonal pairs (Chatterjee et al. 2012;Neumann et al. 2010).
The two widely used PylRS/PylT pairs belong to the archaea Methanosarcinales, M. mazei (Mma) and M. barkeri (Mba), predominantly found in semi-aquatic environments. Recently, a number of new, evolutionary distant, Pyl-encoding archaea have been characterized from the human gut microbiome (Borrel et al. 2014;Borrel et al. 2012;Borrel et al. 2013;Dridi et al. 2012). Here, we explored the utility of the PylRS/PylT pair of Candidatus Methanomethylophilus alvus Mx1201 (Mx1201) in mammalian cells. There were three rationales for this: First, we speculated that proteins of gut-resident archaea might have evolved to optimally perform at human body temperature as opposed to the environmental species that need to be adaptive over a wide temperature range (Gunnigle et al. 2013). Second, Mx1201 PylS gene encodes a smaller protein (31 kDa), which may be easier to express in a heterologous system. PylRS expressed in mammalian cells show predominantly nuclear localization which has been linked to inefficient function (Nikić et al. 2016). Third, divergent evolution from the Methanosarcina PylRS/PylT pairs could manifest in mutual orthogonality. Mutually orthogonal PylRS/PylT pairs would enable incorporation of two distinct non-canonical amino acids (ncAA) using a dedicated tRNA each, within the same host system.
RESULTS AND DISCUSSION
Mx1201 PylRS/PylT pair has unique properties when compared to previously characterized archaeal and bacterial PylRS/PylT pairs
Mx1201 PylS encodes for a 275 amino acid protein, roughly half the size of Mma PylRS (Figure 1a). Mx1201 PylRS is homologous to the C-terminal domain (CTD) of Mma PylRS only, and there is no gene product in the Mx1201 genome with homology to the PylRS N-terminal domain (NTD) found in all previously characterized archaeal PylRS variants (Borrel et al. 2014). The PylRS CTD harbors the catalytic site, binding both Pyl and the anticodon stem of PylT. The NTD has been shown to bind the variable loop region on the opposite side of PylT (Figure 1b) and has been considered essential for aminoacylation activity in vivo (Herring et al. 2007;Suzuki et al. 2017). Notably, bacterial PylS genes encode two separate subunits PylSn and PylSc that structurally correspond to the two domains described above for archaea, suggesting that the complementing roles of PylRS CTD and NTD are conserved across the two kingdoms. In contrast to this, Methanomethylophilus alvus Mx1201 and few related species were the first genomes to be discovered lacking any detectable PylSn homolog (Borrel et al. 2014), indicating that Mx1201 PylRS may have evolved to function entirely without NTD. Mx1201 PylRS and PylT show overall low sequence identity with the Mma PylRS/PylT pair (Figure 1a, b). Mx1201 PylT is one of the most distant homologs of known archaeal PylT (Supplementary Figure 1), has a considerably divergent acceptor stem and appears to have an even further shortened D loop together with a ‘broken’ anticodon stem when compared to Mma PylT (Figure 1b). Given the many questions arising from such unusual features, we set out to characterize Mx1201 activity, specificity and orthogonality in mammalian cells.
Mx1201 PylRS/PylT is a functional amber suppressor, orthogonal to endogenous tRNAs and RS in mammalian cells
Previously, we have employed an efficient plasmid transfection system to direct ncAA incorporation into a GFP reporter protein in mammalian cells (Schmied et al. 2014;Elsässer 2018). Here, we cloned Mma and Mx1201 PylS and PylT genes into a similar plasmid design, expressing PylS from an EF1 promoter and 4xPylT from human U6 or 7SK promoters in tandem repeats (Supplementary Figure 2, Supplementary Table 1). For an initial combinatorial characterization, a plasmid expressing Mma or Mx1201 PylS was cotransfected with a second plasmid expressing four copies of either PylT variant together with the sfGFP150TAG reporter. The Mma PylT used in this study has a base substitution in the anticodon stem, U25C, previously found to increase amber suppression efficiency in E. coli and mammalian cells (Chatterjee et al. 2013;Schmied et al. 2014). Transient transfection was performed in human embryonic kidney (HEK293T) cells. Amber suppression was measured by GFP fluorescence in cell lysates and by western blotting. We used cyclopropene-L-Lysine (CpK, Supplementary Figure 3) as ncAA, which is efficiently incorporated with wildtype Mma PylRS/PylT (Schmied et al. 2014;Elliott et al. 2014).
First, we sought to test if the Mx1201 PylRS/PylT pair was functional in mammalian cells. Expression of Mx1201 PylRS/PylT pair indeed allowed selective incorporation of CpK into the GFP reporter, with 28% yield as compared to a no-stop GFP control (Figure 2a, b; Supplementary Figure 4a). In comparison, the Mma PylRS/PylT pair reached 93% (Figure 2a).
The PylRS/PylT pairs encoded by Mx1201 and Mma differ substantially in their primary sequences. We sought to understand if these PylRS and PylT would cross-react or were in fact non-functional accross species, thus mutually orthogonal. Interestingly, Mma PylRS aminoacylates Mx1201 PylT more efficiently (48% suppression) than Mx1201 PylRS, suggesting optimal enzymatic activity even for the non-cognate PylT. This result also implies that key structural recognition features are conserved between the two distant PylT relatives. In contrary, Mx1201 PylRS did not elicit any measurable amber suppression with Mma PylT. Western blotting and immunostaining using an N-terminal FLAG-tag confirmed expression of Mx1201 PylRS (Figure 2a). Mx1201 PylRS protein levels appeared much higher than for Mma PylRS in our lysates (Figure 2a).
However, it should be noted that Mma PylRS has a distinctive nuclear localization (Supplementary Figure 6), and we only solubilized 50% of the Mma PylRS protein using RIPA buffer (Supplementary Figure 4b). Mx1201 PylRS is soluble and mostly cytosolic (Supplementary Figure 4b, 6). In summary, Mx1201 PylRS is stable and correctly localized in mammalian cells, but does not generate aminoacylation activity equivalent to Mma PylRS. Given the known importance of the PylRS NTD for PylT binding (Herring et al. 2007;Suzuki et al. 2017), we speculate that the Mx1201 PylRS evolved means to partially but not completely compensate for the lack of an NTD.
Further, we tested if additional PylT copies would enhance activity of the Mx1201 PylRS/PylT pair. Indeed, supplying 4xMx1201 PylT on both plasmids raised the amber suppression efficiency to 46% of a no-stop GFP (Figure 2a).
To confirm orthogonality of Mx1201 PylRS/PylT in mammalian cells, we further needed to ensure that Mx1201 PylT is not charged by an endogenous aminoacyl-tRNA synthetase, and that Mx1201 PylRS does not charge other tRNAs with CpK. sfGFP150TAG expression was undetectable in the absence of CpK as judged by fluorescence measurement or western blot (Figure 2a, b). Further, we determined the identity of CpK incorporated into the sfGFP150TAG construct by mass spectrometry (Figure 2c). Vice versa, Mx1201 PylRS does not charge other mammalian tRNAs, as shown by selective SiR-tetrazine reaction with the amber-suppressed GFP and the absence of additional labeled endogenous proteins in whole-cell lysates (Figure 2d).
Together, these results show that the Mx1201 PylRS/PylT pair is functional and orthogonal in mammalian cells.
Mx1201 PylRS can be engineered for expanded substrate specificity
The key advantage of the PylRS/PylT system in mammalian cells over other orthogonal tRNA systems lies in the ability to incorporate structurally diverse ncAAs with useful functions for probing, controlling and engineering proteins in living cells. This is both facilitated by the relative promiscuity of the wildtype PylRS enzyme and the ability to generate active site mutations with expanded substrate repertoire. Exploring the substrate preferences of PylRS/PylT pairs from additional species may further expand the available repertoire of PylRS variants. Having established a new PylRS/PylT pair in mammalian cells, we sought to understand the substrate specificity of Mx1201 PylRS on a small set of functionally interesting ncAAs for mammalian cell biology (Supplementary Figure 3). While Mma PylRS accepted CpK as well as 3’-azibutyl-N-carbamoyl-lysine (AbK) (Figure 3a), Mx1201 PylRS did not incorporate any of the tested ncAAs except CpK (Figure 3b).
Sequence alignment and modeling suggests that, despite overall low sequence identity, the Pyl-binding pocket of Mx1201 PylRS is highly similar to other archaeal and bacterial PylRS homologs (Supplementary Figure 5a). Few exceptions apply, such as Met129 and Val168 at the distal end contacting the pyrrole ring, where most other PylRS, including Mma PylRS, feature a highly conserved Leu and Cys residue, respectively (Supplementary Figure 5b). Thus, there may be subtle variations in the substrate binding pocket underlying the more restricted repertoire of Mx1201 PylRS.
To expand the scope of the Mx1201 PylRS/PylT pair in mammalian cells, we tested the possibility of engineering the Mx1201 PylRS substrate binding pocket. We generated a variant, Mx1201 PylRSY126A, corresponding to a Mma PylRS Y306A mutant. The latter residue caps the Pyl-binding pocket in available PylRS structures and has previously been described to limit PylRS from incorporating ncAAs with longer and/or larger side chains than Pyl (Yanagisawa et al. 2008;Kavran et al. 2007). Mx1201 PylRSY126A has a much reduced activity towards CpK, but intriguingly gained activity (yield 29% of no-stop GFP) for axial trans-Cyclooct-2-ene–Lysine (TCO*K). Comparing this result with the prior PylRS variant described for TCO*K, PylRS-AF (Nikić et al. 2014;Yanagisawa et al. 2008), Mx1201 PylRSY126A has roughly half the activity but dramatically increased specificity for TCO*K over other ncAAs tested.
Mma PylRS requires its NTD for activity towards Mx1201 PylT
Following up on our finding that Mma PylRS accepts Mx1201 PylT (Figure 1a, b), we sought to understand how Mma PylRS recognizes the non-cognate PylT. Specifically, we wondered if Mma PylRS N- and C-terminal domains differentially contributed to cognate Mma PylT or non-cognate Mx1201 PylT binding. We created a new construct, Mma PylRS-CTD, by deleting the first 187 residues of Mma PylRS (Figure 4a). This fragment contains the entire region homologous to the Mx1201 PylRS enzyme (see Figure 1a).
Mma PylRS-CTD was inactive with its cognate PylT (Figure 4b, c). Essentiality of the Mma PylRS NTD has never been explicitly tested in mammalian cells but mirrors observations made in E. coli and in vitro (Herring et al. 2007). Unlike the full-length enzyme, Mma PylRS-CTD also did not elicit measurable activity with Mx1201 PylT (Figure 4b, c). These results show that Mma PylRS activity towards Mx1201 PylT is fully dependent on its N-terminal domain. The results imply two fundamentally different binding modes employed by Mma and Mx1201 PylRS: Mx1201 PylRS has evolved efficient tRNA binding through its catalytic domain, making an N-terminal domain obsolete. This binding mode must be facilitated by specific features of the cognate Mx1201 PylT, since Mx1201 does not accept Mma PylT (Figure 1a). Mma PylRS, on the other hand, employs the canonical NTD interaction with the variable loop region of PylT, a known identity element of PylRS/PylT pairs (Jiang and Krzycki 2012;Ambrogelly et al. 2007). Supporting this hypothesis, the variable loop itself (41CAG43) and adjacent bases of Mx1201 PylT are conserved to their Mma PylT counterparts (Figure 4a).
Generation of an orthogonal PylRS/PylT pair by disrupting NTD interaction of Mx1201 PylT
Above findings provide key prerequisites for the creation of mutually orthogonal PylRS/PylT pairs in mammalian cells: Two evolutionary distant PylRS enzymes (and engineered variants) with partially, but not fully overlapping, substrate specificities, that use distinct surfaces for their recognition of the respective cognate PylT. We hypothesized that rationally designed mutations in the variable loop region of Mx1201 PylT would directly interfere with the non-cognate recognition by Mma PylRS NTD (Figure 5a). Disrupting this interaction would make the Mx1201 and Mma tRNA/aminoacyl-tRNA synthetase pairs mutually orthogonal.
A recent crystal structure of the PylRS-NTD–PylT complex reveals that the recognition relies predominantly on steric constraints that would exclude any more spacious variable loop (Suzuki et al. 2017). We reasoned that introduction of variations to the variable loop analogous to those shown to abrogate PylRS binding and aminoacylation (Jiang and Krzycki 2012;Ambrogelly et al. 2007), would provide candidates for disrupting the interaction between Mx1201 PylT and Mma PylRS.
As a caveat to this simple approach, even isolated base changes may affect folding of the tRNA in unexpected ways, particularly since the short variable loop participates in a tightly packed tertiary core (Nozawa et al. 2009). We chose to test four single base substitutions, C40A, C41A, G43A, G43U, and one insertion C41CA (Figure 5a).
For a more facile generation and screening of Mx1201 PylT mutants, we moved to a three-plasmid expression system where PylRS and sfGFP150TAG reporter were expressed from plasmids without PylT, and PylT was supplied on a third plasmid (Supplementary Figure 2). Wild type and mutant Mx1201 PylT were coexpressed with the same sfGFP150TAG reporter and either Mma PylRS or Mx1201 PylRS (Figure 5b, c). As in previous experiments, Mx1201 PylT was more active with Mma PylRS (89% of no-stop GFP) than its cognate Mx1201 PylRS (42% of no-stop GFP).
None of the Mx1201 PylT mutants exhibited a similar efficiency, suggesting that mutations in the variable loop affected both cognate and non-cognate interactions with Mx1201 PylRS and Mma PylRS, respectively. Nevertheless, C40A, C41A and C41CA mutations preserved measurable activity with Mx1201 PylRS. Of these, C41A retained the highest activity with Mx1201 PylRS (7% of no-stop GFP) while disproportionally reducing Mma PylRS activity (Figure 5b, c). Since we aimed to create a fully orthogonal pair, we focused on C41CA, which showed lower activity with Mx1201 PylRS but lost all detectable activity with Mma PylRS even by sensitive western blot (Figure 5c). Moving the Mx1201 PylTC41CA mutant back to our efficient two-plasmid transfection system (Supplementary Figure 2), we were able create robust amber suppression with Mx1201 PylRS, and confirm orthogonality to Mma PylRS (Figure 5d, e; for full panel see Supplementary Figure 7).
In conclusion, we we have created a new tRNA/aminoacyl-tRNA synthetase pair, Mx1201 PylS/Mx1201 PylTC41CA, that is orthogonal in mammalian cells, and also mutually orthogonal to the Mma PylRS/PylT pair.
Site-specific incorporation of two distinct ncAAs using two orthogonal PylRS/PylT pairs
Next, we aimed to implement dual site-specific protein modification at independent sites and with distinct ncAAs in mammalian cells using the two orthogonal PylRS/PylT pairs. This first required to further modify one pair to recode another stop codon. We chose ochre (TAA) because it is only marginally more abundant than amber in mammalian cells. We thus created Mma PylTUUA, where the anticodon was changed from CUA to UUA. We used a fluorescent reporter, sfGFP102TAG+150TAA, containing two stop codons, 102TAG and 150TAA.
To incorporate two distinct ncAAs, the two orthogonal PylRS must each incorporate one of the ncAAs with high selectivity (Figure 6a). Since we have shown that wild type Mx1201 PylRS and the engineered variant PylRSY126A both have narrow substrate specificities, they can be combined in many unique combinations with known Mma PylRS variants evolved for specific ncAAs, e.g. photocaged lysine (PcK), acetyl-lysine (AcK), diazirine lysine (AbK) (Neumann et al. 2008;Gautier et al. 2010;Ai et al. 2011).
Here, we chose to incorporate AcK and TCO*K into the same protein, GFP, in response to TAA and TAG stop codons, respectively: we created a pair with Mma PylTUUA and AcKRS (Neumann et al. 2008) and combined it with the Mx1201 PylRSY126A/Mx1201 PylTC41CA pair in the same cell (Figure 6a). Indeed, we were able to observe production of GFP in cells transfected with the two PylRS/PylT pairs and the sfGFP102TAG+150TAA reporter in the presence of both ncAA, but not with either individually or none (Figure 6b, c). Thus, we have shown that the two orthogonal PylRS/PylT pairs can be employed together in the same cell to site-specifically incorporate two distinct ncAAs.
CONCLUSIONS
We have set out to identify new orthogonal tRNA/aminoacyl-tRNA synthetase pairs in mammalian cells. Exploring the function of a previously uncharacterized distant homolog of the Mma PylRS/PylT pair from Methanomethylophilus alvus Mx1201, we found that the Mx1201 PylRS/PylT pair is active and orthogonal in mammalian cells. Mx1201 PylRS is also orthogonal to Mma PylT. We show that the Mx1201 PylRS enzyme can be engineered to expand its ncAA substrate spectrum, creating a new variant Mx1201 PylRSY126A that can efficiently incorporate TCO*K. Mx1201 PylRS is a small enzyme (31 kDa) that lacks the N-terminal domain present in all previously characterized archaeal PylRS proteins. Mma PylRS can accept the non-cognate Mx1201 PylT as substrate, and intriguingly we find that this is entirely dependent on its NTD. This indicated that Mx1201 and Mma PylRS rely on different identity determinants of PylT. We used this knowledge to rationally design a series of mutant Mx1201 PylT with modified variable loop region in order to selectively disrupt its interaction with Mma PylRS. We identify several Mx1201 PylT mutants that retain their cognate interaction with Mx1201 PylRS but reduce or abrogate non-cognate recognition by Mma PylRS. We selected one mutant, Mx1201 PylTC41CA for further experiments. We show that the Mx1201 PylS/ PylTC41CA pair is orthogonal to Mma PylRS/PylT. Combined in the same cell, we show that the two pairs can introduce two different ncAAs in response to two distinct stop codons. Our findings shed light into a new clade of PylRS enzymes with unexpected properties, functionally divergent from the previously studied archaeal and bacterial system. Our study agrees with a recent first characterization of the Mx1201 PylRS/PylT pair in E. coli published during the preparation of this manuscript (Willis and Chin 2018). Notably, Mx1201 PylRS/PylT pair exceeds the activity of Mma PylRS/PylT pair in E.coli. Chin and Willis also used a similar rational for creating orthogonal PylRS/PylT pairs and showed their excellent activity in E.coli. Intriguingly their directed evolution approach to identify most highly active orthogonal Mx1201 PylTs yields additional candidates for mammalian cells. Our work expands the repertoire of mutually orthogonal tools for genetic code expansion in mammalian cells and provides the basis for advanced in vivo protein engineering applications for cell biology and protein production.
MATERIALS AND METHODS
DNA constructs
The sfGFP 150L control reporter construct has been described previously (Schmied 2014). A series of plasmids for amber suppression (pAS) was created based on PB510B-1 (System Biosciences) piggybac plasmid (Table 1 and Supplementary Figure 2). pUC-based plasmids were used to express tRNA from single copy genes (Table 2 and Supplementary Figure 2). All DNA constructs were verified by Sanger sequencing.
Cell culture and Transfection
HEK293T cells were maintained in adherent culture at 37°C and 5 %CO2 atmosphere in Dulbecco’s modified Eagle’s medium (DMEM) (AqDMEMTM high glucose, Sigma) supplemented with 10% FBS. For transfection 1.5 - 2.0×105 HEK293T cells were seeded per well in 24-well plates 1d before transfection. Cells were transiently transfected with TransIT®-LT1 (Mirus) following manufacturer’s instructions. ncAAs were added at the time of transfection as indicated and transfected cells harvested 24h or 48h post transfection.
Amino acids
Non-canonical amino acids (ncAAs) used in this study are summarized below. To incorporate ncAA into proteins, amino acid working solutions were prepared from 100 mM stock solutions and added to the cultured cells together with the transfection mixture.
Mass Spectrometry
HEK293T cells were transfected and cultured in presence of ncAA for 72h. After cell lysis in RIPA buffer with added cOmplete protease inhibitor (Roche), the insoluble fraction was removed by centrifugation. GFP was captured on GFP-Trap_M magnetic beads (ChromoTek) and eluted with 1% acetic acid.
Mass spectrometric analysis was carried out at the Proteomics Biomedicum core facility, Karolinska Institutet, Stockholm. Intact protein mass analysis was performed using a TriVersa NanoMate chip-based electrospray device (Advion, Ithaca, NY) coupled to the LTQ Velos Orbitrap Elite (Thermo Scientific, Bremen, Germany). The ChipSoft Manager software was used to control the TriVersa NanoMate, while data was acquired directly from the Tune software of the mass spectrometer. The NanoMate delivered 2 μL of sample solution to the tip engaged with the back of the ESI chip and nano-spray ionization was initiated applying 1.9 kV and 0.8 psi gas pressure. The mass spectrometer was operated in positive ion mode with activated protein mode settings. MS data was collected in full scan mode (m/z 500-2000) with a resolution of 100,000 at m/z 400. Each scan comprises 1 microscan. The mass spectra shown are comprised of approximately 20 scans. Automatic gain control (AGC) was used to accumulate sufficient ions for analysis targeting 3×107 ions in a maximum fill time of 200 ms. Data were analyzed using the Protein Deconvolution v3.0 software (Thermo Scientific) to calculate monoisotopic masses.
Quantification of GFP expression
Transfected HEK293T cells were grown in presence of ncAA as indicated for 24h or 48h. Cells were lysed in RIPA buffer with 1× cOmplete protease inhibitor (Roche), the insoluble fraction was removed by centrifugation. GFP bottom fluorescence of an aliquot was measured in Tecan Infinity M200 pro platereader (excitation 485 nm, emission 518 nm). Fluorescence measurements were normalized to total protein content of each sample as determined by Pierce BCA assay kit (Fisher Scientific) on the same sample.
Live-cell imaging for GFP expression
GFP expression was visualized by imaging in a ZOE™ Fluorescent Cell Imager (BioRad).
Western Blot
Equal amounts of protein, as determined by Pierce BCA assay (Fisher Scientific) were separated on 4-20% Tris-glycine gels (BioRad) and transferred to nitrocellulose membranes. Expression of the sfGFP reporter and aminoacyl-tRNA synthetases was confirmed by immunoblotting with antibodies against GFP (Santa Cruz, sc-9996), FLAG-HRP (Sigma, A8592), β-Actin (cell signaling) and corresponding secondary HRP-conjugated antibodies when needed (BioRad).
Immunostaining and fluorescence microscopy
Transfected cells were cultured in presence of ncAA for 24h. Before fixation (4% formaldehyde) and permeabilization (0.1% triton) the ncAA was removed for 8h. Samples were blocked in 2% BSA in TBS-T and subsequently incubated in presence of 0.5 mM SiR-tetrazine (Spirochrome). After washing with TBS-T samples were incubated with primary antibodies mouse anti-GFP (B-2, Santa Cruz #9996) and rabbit anti-FLAG (D6W5B, Cell Signaling #14793) and subsequently incubated with secondary antibodies anti-mouse Alexa Fluor 488, anti-rabbit Alexa Fluor 555 (Life Technologies) and DAPI (Sigma-Aldrich). After washing, cells were imaged on a Nikon eclipse Ti2 inverted widefield microscope, using a 20× 0.75 NA objective.
AUTHOR CONTRIBUTIONS
SJE and BM conceived and planned experiments. BM and JH carried out experiments and analyzed data. LL performed immunofluorescence microscopy and analyzed data. BM, JH and SJE prepared figures and wrote manuscript.
FUNDING INFORMATION
Research was funded by Karolinska Institutet SFO Molecular Biosciences, Vetenskapsrådet (2015-04815), H2020 ERC-2016-StG (715024 RAPID), Ming Wai Lau Center for Reparative Medicine, Ragnar Söderbergs Stiftelse.
ACKNOWLEDGEMENTS
Mass Spectrometry service was provided by Proteomics Biomedicum core facility, Karolinska Institutet, Stockholm. We thank Akos Vegvari for data analysis and advice.
We thank the Jiri Bartek lab for access to Tecan Infinity M200 Pro plate reader and Nikon eclipse Ti2 microscope.
Footnotes
↵* simon.elsasser{at}scilifelab.se