Genetic code expansion in mammalian cells: A plasmid system comparison

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Abstract

Genetic code expansion with unnatural amino acids (UAAs) has significantly broadened the chemical repertoire of proteins. Applications of this method in mammalian cells include probing of molecular interactions, conditional control of biological processes, and new strategies for therapeutics and vaccines. A number of methods have been developed for transient UAA mutagenesis in mammalian cells, each with unique features and advantages. All have in common a need to deliver genes encoding additional protein biosynthetic machinery (an orthogonal tRNA/tRNA synthetase pair) and a gene for the protein of interest. In this study, we present a comparative evaluation of select plasmid-based genetic code expansion systems and a detailed analysis of suppression efficiency with different UAAs and in different cell lines.

Introduction

Proteins carry out the essential processes of life through their diverse structures and functions. These properties arise from the 20 natural amino acids and their extensive post-translational modifications (PTMs), such as methylation, glycosylation, and phosphorylation. Despite the diversity and variability of natural proteins’ functions, the natural amino acids have limited chemical functionalities and reactivity compared to the repertoire of synthetic organic chemistry. These limitations have been overcome through pioneering work by Schultz and others on expanding the genetic code for the incorporation of customizable and chemically unique unnatural amino acids (UAAs).1, 2 UAA mutagenesis in mammalian cells has facilitated the study of biological events at the molecular level, such as proximity-based protein–protein interactions in living organisms3, 4, 5; activation of protein function using small molecules6, 7, 8 and light9, 10, 11, 12, 13, 14; and introduction of various PTMs, including phosphorylation,15, 16 acetylation,17 crotonylation,18 and SUMOylation.8 Genetic code expansion has also been achieved in animals.19, 20 Apart from providing a better understanding of the molecular mechanisms of protein functions, this technique has also been applied to the development of therapeutics, including site-specific antibody-drug conjugates (ADCs),21, 22, 23, 24, 25 controllable Chimeric Antigen Receptor T (CAR-T) cells,26, 27, 28 stabilized29, 30 and spatiotemporally controllable31 adeno-associated viruses, and attenuated live vaccines.32, 33

UAA mutagenesis employs orthogonal aminoacyl-tRNA synthetase (aaRS)-tRNACUA pairs to suppress amber stop codons (UAG), resulting in the incorporation of the UAA. This strategy requires the delivery of the following components into host cells: 1) the UAA, 2) the gene encoding an engineered aaRS mutant for the desired UAA, 3) the gene for the tRNACUA, and 4) the gene for the target protein carrying the amber codon at the desired position.

While the production of sufficient protein-of-interest (POI) with the UAA incorporated is crucial, the efficiency of UAA incorporation is often limited in mammalian cells34 and animals,20 frequently requiring optimization. Numerous factors influence the efficiency of UAA mutagenesis, some of which often cannot be changed, such as the position of UAA insertion and the chemical structure of the UAA. However, other parameters can be optimized to improve protein yields, such as UAA concentration, aaRS and tRNACUA expression levels, POI mRNA transcription levels, and the aaRS’s catalytic efficiency. It should also be noted that the sequence context of the nonsense codon can be altered within the limits of silent mutations for improved suppression efficiency, which is outside the scope of this work.35

Among the aaRS-tRNA pairs used for UAA mutagenesis, the pyrrolysyl-tRNA synthetase/tRNACUA (PylRS/PylT) pair has risen to prominence as the most widely applied.36 This system is advantageous because the pair is orthogonal in both mammalian cells and E. coli hosts, allowing rapid testing and optimization in E. coli before moving into higher cells and organisms. Further, PylRS is able to recognize chemically diverse UAAs, and it does not have a natural substrate in bacterial and mammalian cells.37 Among these substrates, we have chosen to focus on N-allyloxycarbonyl-lysine (AOK) and hydroxycoumarin lysine (HCK). The former is a well-validated positive control for several PylRS variants (including wild-type) as well as handle for bioconjugation via photoinitiated thiol-ene reactions,38 Pd-catalyzed deallylation, and inverse electron-demand Diels-Alder reactions. The latter is a photocaged lysine that has been incorporated into several proteins in mammalian cells and zebrafish, for example enabling the activation of signaling pathways and DNA manipulation using light.39, 40, 41 The conclusions drawn from our work are expected to generalize to many other UAAs.

We utilized an established mCherry-TAG-EGFP fusion protein as a reporter to gauge the incorporation efficiency13 of both AOK and HCK by HCKRS (the Y271A-L274M mutant of PylRS)40 using different vectors and cell types. When cells translate this protein, mCherry is first produced, then the ribosome reaches the UAG codon. Here, tRNACUA-AOK or tRNACUA-HCK adds the UAA to the growing polypeptide chain, allowing for subsequent EGFP translation. Our results with this reporter demonstrate that any of the aforementioned factors (concentrations of UAA, expression levels of aaRS, copy numbers of tRNACUA cassettes, promoter of the POI, and aaRS variants) can be the limiting factor under different circumstances.

Section snippets

Results and discussion

Here, we conducted a comparative study of four commonly used plasmid systems for UAA incorporation in mammalian cells (Fig. 1): 1) the pAG dual-plasmid system originally developed by the Chin Lab13 and re-engineered by us (①),40 2) a new pcDNA dual-plasmid system developed by our lab(②), 3) the pE323/363 dual-plasmid system developed by the Chin Lab (③ and ④),34 and 4) the pEA single plasmid system developed by the Arbely Lab (⑤-⑦).42

We tested multiple versions of these systems to dissect the

Conclusion

Overall, of these commonly used platforms for UAA incorporation in mammalian cells, we conclude that the pE323/pE363 two-plasmid system ( and ) delivers the highest incorporation efficiency across different UAA species and cell types, in particular when using a PylRS chimera with an optimized N-terminal domain (). In this system, one plasmid contains a (U6-PylT)4 cassette and PEF1α-PylRS, and the other contains the PEF1α-POI and an additional (U6-PylT)4 cassette. The PylRS-PylT plasmid from

Mammalian cell culture and transfection

NIH3T3 and HEK293T cells were acquired from ATCC and maintained in DMEM (Fisher) with 10% FBS (GE Healthcare) without antibiotics and incubated at 37 °C, 5% CO2. All experiments were performed in triplicate.

For imaging purposes, cells were seeded in poly-d-lysine (70,000–150,000 kDa, MP Biomedicals)-treated 96-well plates (Greiner Bio-One) at 20,000 cells/well. When cell density reached ~ 90%, growth media was changed to 90 μL fresh DMEM with 10% FBS. To prepare the transfection reagent, 100 ng

Declaration of Competing Interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Acknowledgement

We dedicate this publication to Dr. Peter G. Schultz for being awarded the 2019 Tetrahedron Prize for Creativity. This research was supported by the US National Institutes of Health (R01GM132565) and the National Science Foundation (CBET-1603930). We thank the Chin Lab for the pmCherry-TAG-EGFP plasmid, the pE323_wildtype MmPylRS_4xPylT plasmid, and the pE363_mCherry-TAG-EGFP_4xPylT plasmid. We thank the Arbely Lab for the pEA_4xPylT plasmid.

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