Ser/Leu-swapped cell-free translation system constructed with natural/in vitro transcribed-hybrid tRNA set

doi:10.21203/rs.3.rs-3330085/v1

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Ser/Leu-swapped cell-free translation system constructed with natural/in vitro transcribed-hybrid tRNA set

https://doi.org/10.21203/rs.3.rs-3330085/v1

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The Ser/Leu-swapped genetic code can act as a genetic firewall, mitigating the biohazard risks arising from horizontal gene transfer in genetically modified organisms. Our prior work demonstrated the orthogonality of this swapped code to the standard genetic code using a cell-free translation system comprised of 21 in vitro transcribed tRNAs. To advance this system for protein engineering, we introduced a natural/in vitro transcribed hybrid tRNA set. This set combined natural tRNAs from Escherichia coli (excluding Ser, Leu, and Tyr) and in vitro transcribed tRNAs, encompassing anticodon-swapped tRNA^Ser_GAG and tRNA^Leu_GGA. This approach reduced the requisite in vitro transcribed tRNAs from 21 to a mere 4. In this optimized system, the production of a model protein, super-folder green fluorescent protein, increased to 3.5-fold. With this hybrid tRNA set, the Ser/Leu-swapped cell-free translation system will stand as a potent tool for protein production for reduced biohazard concerns in future biological endeavors.

Biochemical Research Methods

Genetic code

tRNA

Translation

Bacterial synthesis of proteins using a genetically modified organism (GMO) transformed with a gene from pathogenic organisms poses a biohazard risk. A hazardous gene that leaks from the GMO could be transferred to other natural organisms through horizontal gene transfer^1–3. Although a cell-free translation system presents a useful solution for synthesizing such proteins, gene leaks could still occur during the preparation of the genes using GMO.

To address this issue, in our previous article published in 2020⁴ (Figure S1), we introduced the Ser/Leu-swapped genetic code. This genetic code allows for the synthesis of a protein without requiring the preparation of a hazardous gene. In this genetic code, Ser is assigned to Leu codons and vice versa, by utilizing chimeric tRNA^Ser_GAG and tRNA^Leu_GGA, both of which have swapped anticodons. Genes encoded by the Ser/Leu-swapped genetic code are not hazardous to natural organisms as they only produce Ser/Leu-swapped inactive proteins in nature.

However, the initial proof-of-concept version of the Ser/Leu-swapped cell-free translation system required the preparation of 21 tRNAs through in vitro transcription. This made the cell-free translation system impractical for protein synthesis. Additionally, in vitro transcribed tRNAs lack post-transcriptional modifications, whereas natural tRNAs extracted from Escherichia coli (E. coli) were reported to have, on average, 7.7 post-transcriptional modifications per molecule⁵. These modifications play diverse roles, including the altering the codon-amino acid correspondence^{6, 7}, modulating the decoding efficiencies of synonymous codons^8–10, promoting proper tRNA folding^11–16, and facilitating the efficient recognition of tRNAs by IleRS, GluRS, and LysRS^17–19. In fact, the production of proteins dropped to 20% when replacing the natural tRNA extract with the IVT tRNA set.

In this study, we created natural/IVT-hybrid tRNA set. This set consists of tRNA^Ser, tRNA^Leu, and tRNA^Tyr-deficient natural tRNAs extracted from E. coli, alongside tRNA^Phe, tRNA^Tyr, chimeric tRNA^Ser_GAG, and chimeric tRNA^Leu_GGA synthesized through in vitro transcription. Utilizing this hybrid tRNA set, we established a Ser/Leu-swapped cell-free translation system (Fig. 1) that achieved protein production efficiency comparable to a translation system using natural tRNAs.

Construction of the natural/ in vitro transcribed (IVT)-hybrid tRNA set

E. coli has 37 tRNAs that decode 61 codons into 20 proteinogenic amino acids. Among these, tRNA^Ser, tRNA^Leu, and tRNA^Tyr are longer (85–93 bases) than the other tRNAs (74–77 bases) (Fig. 2A). This characteristic allows us to prepare a tRNA^Ser and tRNA^Leu deficient natural tRNA set without having to isolate each individual tRNA.

Initially, we analyzed the E. coli tRNA extract using denaturing PAGE containing urea (Fig. 2A, lane 1). The mobility of band I was consistent with that of IVT tRNA^Ser, tRNA^Leu, and tRNA^Tyr (Fig. 2A, lanes 3–5). Thus, we inferred that this band represents the natural tRNA^Ser, tRNA^Leu, and tRNA^Tyr. We harvested the tRNAs in band II (Fig. 2A, lane 2) and defined this tRNA mixture as the ∆SLY natural tRNA set.

Subsequently, we prepared IVT tRNA^Tyr, chimeric tRNA^Ser_GAG, and chimeric tRNA^Leu_GGA using T7 RNA polymerase. We then combined these with the ∆SLY natural tRNA set to assemble the natural/IVT-hybrid tRNA set for the Ser/Leu-swapped genetic code, which we termed the hybrid-SL tRNA set (Fig. 2B). Similarly, we assembled the natural/IVT-hybrid tRNA set for the standard genetic code (hybrid-Std tRNA set) using the IVT tRNA^Ser and tRNA^Leu instead of chimeric tRNAs. We also assembled a tRNA set with 21 IVT tRNAs for the Ser/Leu-swapped genetic code, termed the IVT-SL tRNA set. Using one of these three tRNA sets, we prepared reconstituted cell-free translation systems for further study (Figure S2).

Synthesis of peptides in a cell-free translation system with the hybrid-Std tRNA set

To verify the accurate decoding of codons in the cell-free translation system using the hybrid-Std tRNA set, we first assessed the synthesis of model peptides (Met-Tyr-Tyr-Tyr-Xaa-Asp-Asp-Arg-Asp, where Xaa represents 17 types of amino acids; Fig. 3A). The matrix assisted laser desorption/ionization-time of flight mass spectrometer (MALDI-TOF-MS) analysis demonstrated that all peptides were synthesized in the cell-free translation system (Fig. 3B top and Figure S3A; calculated and observed masses are listed in Tables S1 and S2). Tricine SDS-PAGE analysis followed by autoradiography ([¹⁴C]-Asp labeling) showed comparable amounts of peptides synthesized across all samples (Fig. 3C and Figure S3B). These results suggest that the natural tRNA species corresponding to 17 natural amino acids in the ΔSLY natural tRNA set accurately decode Xaa codon. Tyr insertion was unexpectedly observed for peptides with Glu, Phe, Trp, and Lys. This anomaly might be due to translational slippage on repeated Tyr codons preceding the Xaa codon. Various translational slippage, such as − 1, − 4, and + 2 frameshift at the NNA-AAC-AAG sequence in an E. coli cell-free translation system²⁰, as well as + 2 frameshift at the GUG-UG sequence and + 6 ribosome hopping at the GUG-UGA-GUU sequence in E. coli²¹, have been reported. In addition, the depletion of an aminoacyl-tRNA that corresponds to an A-site codon can increase the − 1 frameshifting in an E. coli cell-free translation system²². This suggests that the unexpected insertion of Tyr observed in the mass spectra might be due to the low concentration of aminoacyl-tRNAs of Glu, Phe, Trp, and Lys (vide infra).

Accuracy of Ser/Leu-swapping in a cell-free translation system with the hybrid-SL tRNA set

The presence of natural tRNA^Ser and tRNA^Leu in the IVT-SL tRNA set could compromise accurate translation in the Ser/Leu-swapped cell-free translation system, leading to the synthesis of proteins that contain both Ser and Leu at the same positions. While the PAGE analysis indicated no detectable amount of natural tRNA^Ser and tRNA^Leu in the ∆SLY natural tRNA set (Fig. 2A, lane 2), we examined the accuracy of Ser/Leu-swapping by translating model peptides (Met-Tyr-Tyr-Tyr-Xaa-Asp-Asp-Arg-Asp, where Xaa represents amino acid at UCU or CUU codons; Fig. 3A).

Using a cell-free translation system with the hybrid-SL tRNA set, we synthesized the model peptides with Ser or Leu at the Xaa position. MALDI-TOF-MS analysis confirmed that Ser and Leu were accurately assigned to their respective swapped codons in the Ser/Leu-swapped genetic code without mutual contamination (Fig. 3B middle and Table S1). Tricine SDS-PAGE analysis further supported this finding as translated products formed a single intense band for each mRNA (Fig. 3C).

To assess the importance of excluding natural tRNA^Ser and tRNA^Leu from the E. coli tRNA extract, we additionally tested a mix of intact E. coli tRNA extract with chimeric tRNA^Ser_GAG and tRNA^Leu_GGA. The mixture of peptides with Ser and Leu were detected in both MALDI-TOF-MS (Fig. 3B bottom and Table S1) analysis and tricine SDS-PAGE (Fig. 3C). These results indicated that the excluding the natural tRNA^Ser and tRNA^Leu from E. coli tRNA extract is crucial for the accurate Ser/Leu-swapping in the cell-free translation system.

Optimization of the hybrid tRNA set for translating a protein

We next attempted to synthesize a model protein in the cell-free translation system using the hybrid-Std tRNA set. We employed standard sfGFP gene encoding superfolder GFP (sfGFP) according to the standard genetic code and assessed the sfGFP production by a spectrofluorometer (Figure S5A, Std-22C). Unexpectedly, the fluorescence intensity of sfGFP synthesized using hybrid-Std tRNA set was only 4% compared to synthesis using E. coli tRNA extract (Figure S6).

Considering the possibility that the concentration of certain natural tRNA(s) may have been reduced during the PAGE purification process, we attempted to identify these tRNA(s) by adding IVT tRNAs. We divided 18 IVT tRNAs into four groups (Groups A–D) and assessed whether the sfGFP fluorescence intensity improved upon adding each group of tRNAs. Notably, only Group A tRNAs (comprising tRNA^Phe, tRNA^Lys, tRNA^Trp, tRNA^Cys, and tRNA^Ala) significantly enhanced sfGFP production (Figure S6). Then, we added each tRNA from Group A to the cell-free translation system. We found that only the addition of tRNA^Phe increased the production of sfGFP to approximately 78% of that from the natural tRNA extract (Fig. 4). The denaturing PAGE analysis revealed a smeared band corresponding to IVT tRNA^Phe (Fig. 2A, lane 6), suggesting partial removal of tRNA^Phe during the PAGE purification. Therefore, we decided to include IVT tRNA^Phe in the hybrid tRNA sets for subsequent experiments.

Ser/Leu-swapped synthesis of a model protein using the hybrid-SL tRNA set

To study the synthesis of the model protein sfGFP in the Ser/Leu-swapped cell-free translation system, we prepared a Ser/Leu-swapped sfGFP gene encoded according to the Ser/Leu-swapped genetic code (Figure S5A, SL-22C). Native PAGE analysis showed that the amount of active sfGFP in the cell-free translation system with the hybrid-SL tRNA set was comparable to that with the E. coli tRNA extract (Fig. 5, lane 4 vs. 1). Furthermore, the amount of active sfGFP was significantly improved to 3.5 times compared to that with the IVT-SL tRNA set (Fig. 5, lane 4 vs. 5). This result clearly indicates that the cell-free translation system with the hybrid-SL tRNA set is useful for in vitro protein production.

We also studied the orthogonality of the Ser/Leu-swapped genetic code and the standard genetic code by translating the protein using mismatched tRNA set/gene combinations: i.e., the Ser/Leu-swapped sfGFP gene with E. coli tRNA extract and the standard sfGFP gene with the hybrid-SL tRNA set. Indeed, no active sfGFP was detected in either cell-free translation system (Fig. 5, lanes 2 and 3). This is because the translation products synthesized by incorrect combinations contained 30 mutations (10 mutations of Ser to Leu and 20 mutations of Leu to Ser) (Figure S5B); hence, 13% of the residues in the sfGFP (comprising 239 residues) were mutated. Autoradiography of the same gels also showed that proteins with different mobilities from sfGFP were synthesized in both cell-free translation systems with mismatched tRNA set/gene combinations (Fig. 5, lanes 6–10).

Comparison of hybrid-SL tRNA and IVT-SL tRNA sets for translating genes encoded by 22 codons or 47 sense codons

Using the IVT-SL tRNA set, only 35 of the 61 sense codons (Figure S2) are available to encode a protein, as the tRNA set consists of 21 IVT tRNAs. In contrast, 53 of the 61 sense codons are available to encode a protein when using the hybrid-SL tRNA set, as the tRNAs for 16 standard amino acids (excluding Ser, Leu, Tyr, and Phe) were isolated from the E. coli tRNA extract (Figure S2).

In the aforementioned study, we used a Ser/Leu-swapped sfGFP gene containing 22 sense codons (Figure S5A, SL-22C). To verify codon availability, we prepared a Ser/Leu-swapped sfGFP gene containing 47 sense codons (excluding rare codons) (Figure S5A, SL-47C). The synthesis of inactive proteins in a cell-free translation system using natural tRNA extract from this gene demonstrated the orthogonality of the Ser/Leu-swapped genetic code with 53 sense codons against the standard genetic code (Figure S8).

In the cell-free translation system with the hybrid-SL tRNA set, a similar amount of active sfGFP was synthesized from both genes (Fig. 6, lanes 1, 2, 5, and 6), suggesting that at least 47 sense codons are available to encode a protein. In contrast, while sfGFP was synthesized from the Ser/Leu-swapped sfGFP gene containing 22 sense codons (Fig. 6, lanes 4 and 8), none was produced from the gene containing 47 sense codons (Fig. 6, lanes 3 and 7) in the cell-free translation system using the IVT-SL tRNA set. This is attributed to the IVT-SL tRNA set’s lack of tRNAs to decode 14 codons, representing 26% of the coding sequence, in the SL-47C_sfGFP gene.

Recent studies have reported that the choice of the nonsynonymous codons affects the accuracy of an amino acid incorporation²³ and a protein folding through the control of the translation rate^{24, 25} for certain proteins. Hence, the cell-free translation system with the hybrid-SL tRNA set could be invaluable for designing a gene.

In summary, we successfully developed a cell-free translation system with the Ser/Leu-swapped genetic code, employing the natural/IVT-hybrid tRNA set. This hybrid tRNA set enhanced the sfGFP production, achieving a 3.5-fold increase compared to the previously reported IVT-SL tRNA set. This not only improves translation efficiency but also reduces the associated effort needed to prepare the tRNA set for the Ser/Leu-swapped genetic code. Importantly, the majority of the natural tRNAs were retained in the natural/IVT-hybrid tRNA set, making 53 out of 61 sense codons available for designing Ser/Leu-swapped genes. Our study also demonstrated that proteins translated from Ser/Leu-swapped sfGFP genes were inactive when translated in the cell-free translation system with the standard genetic code. This finding underscores the reduced risk of gene leakage to natural organisms when employing Ser/Leu-swapped genes. The concept of an orthogonal genetic code, especially one that with amino acid-swapping has garnered interest^{26, 27} since our report in 2020⁴. Recently, the successful prevention of phage replication, facilitated through partial Ser/Leu-swapping by expressing chimeric tRNA^Leu_YGA in the codon compressed E. coli was demonstrated²⁸. In addition to in vivo approaches from other groups, our Ser/Leu-swapped cell-free translation system offers promising avenues to mitigate future biohazard risks.

Materials

Natural tRNA extract derived from E. coli MRE600 strain was purchased from Merck (Tokyo, Japan). Synthetic DNAs containing Std-22C_sfGFP and SL-22C_sfGFP genes were purchased from Integrated DNA Technologies (Tokyo, Japan). A plasmid containing the SL-47C_sfGFP gene was purchased from Eurofins Genomics (Tokyo, Japan). Primers were purchased from FASMAC (Kanagawa, Japan). mRNAs encoding 17 model peptides and 21 IVT tRNAs were prepared following the procedure described in our previous work⁴.

Preparation of sfGFP mRNAs

Synthetic Std-22C_sfGFP and SL-22C_sfGFP genes were cloned into the pUC19 vector. DNA templates for mRNA coding sfGFP were amplified by PCR (1000 µL) under the following conditions: 1 × PfSH buffer [10 mM Tris-HCl pH 8.4, 100 mM KCl, 0.1% (v/v) Triton X-100, 2 mM MgSO₄], 2% DMSO, 0.2 mM of each dNTP, 0.375 µM T7Aex.F24 (5′-GGCGTAATACGACTCACTATAG-3′), 0.375 µM ASsfGFP.R20 (5′-TGACTAAGAATCCAGTATGC-3′), plasmid containing sfGFP genes (Std-22C, SL-22C, or SL-47C; see Figure S5), and 2 nM Pfu-S DNA polymerase. DNA was amplified by 12 cycles of 95°C for 20 s, 50°C for 20 s, and 72°C for 1 min. The resultant DNA was purified using phenol/chloroform extraction followed by isopropanol precipitation. It was then dissolved in 10 mM Tris-AcOH pH7.8 (100 µL). mRNAs were produced by run-off in vitro transcription (1000 µL) using the following conditions: 1 × T7 buffer [40 mM Tris-HCl pH 8.0, 1 mM spermidine, 0.01% (v/v) Triton X-100], 10 mM DTT, 30 mM MgCl₂, 5 mM of each NTP, template DNA (100 µL), and 0.12 µM T7 RNA polymerase at 37°C overnight. Transcribed mRNAs were purified through phenol/chloroform extraction followed by isopropanol precipitation and were dissolved in ultrapure water. The concentrations of mRNAs were determined using a NanoDrop-spectrophotometer ND-1000.

Preparation of ΔSLY natural tRNAs

A denaturing PAGE gel was prepared with acrylamide/bisacrylamide (37.5:1, 10%), 6 M urea, and TBE (44.5 mM tris·borate pH 8.0, 2 mM EDTA) in a large gel cassette [height = 15 cm, width = 15 cm, gel thickness = 2 mm, 1 well (depth = 2 cm, width = 5 cm)]. A loading sample containing natural tRNA extract (200 µL; 50 mg/mL tRNA extract, 4 M urea, 12.5 mM EDTA pH 8.0) was applied to the gel. Electrophoresis was carried out at 300 V for 100 min at room temperature. Band II (Fig. 2A) was cut out, then crushed using a mortar and a pestle. The crushed gel was resuspended in 15 mL of 0.3 M NaCl, and the solution was shaken over night at room temperature to elute the tRNAs. The gel debris was removed by centrifugation, and twice volume of ethanol was added to the supernatant. The solution was incubated at − 20°C for 30 min and then centrifuged (4°C, 13000 rpm, 15 min). After removing the supernatant, the precipitate was dissolved in the 1 mL of ultra-pure water. After filtering through a 45 µm syringe filter (Millipore) and treating with phenol/chloroform, tRNAs were pelleted by ethanol precipitation. The resultant pellet was air-dried and redissolved in the 100 µL of ultra-pure water. This tRNA mixture was termed the ΔSLY natural tRNA set. Both the ΔSLY natural tRNA set and the 10 mg/mL intact natural tRNA extract were analyzed using denaturing PAGE (6 M urea, 10% acrylamide). The gel was stained with ethidium bromide, imaged using Gel Doc EZ system (Bio-Rad), and analyzed with Image Lab software (Bio-Rad). The ΔSLY natural tRNA set stock solution was prepared by adding ultra-pure water to achieve the same concentration of band II tRNAs as in the 10 mg/mL intact natural tRNA extract.

Preparation of tRNA stock solutions

Apart from the ΔSLY natural tRNA set stock solution and the 10 mg/mL intact natural tRNA extract, the IVT tRNA mixes for both the standard genetic code (tRNA^Ser, tRNA^Leu, and tRNA^Tyr; 100 µM each) and the Ser/Leu-swapped genetic code (chimeric tRNA^Ser_GAG, chimeric tRNA^Leu_GAA and tRNA^Tyr; 100 µM each) were prepared. For optimizing the hybrid tRNA set, four IVT tRNA stock solutions (Group A, B, C, and D; 50 µM for each tRNA) in Figure S6, and 100 µM of IVT tRNAs (tRNA^Phe, tRNA^Lys, tRNA^Trp, tRNA^Cys, and tRNA^Ala) in Fig. 4 were also prepared. These stock solutions were heated at 95°C for 5 min and then cooled to 25°C to refold tRNAs.

Cell-free translation

Cell-free translation systems for hybrid tRNA sets were prepared using a procedure similar to that described in our previous work²⁹. The translation mixture (5 µL) was prepared by adding 0.75 µL of the ΔSLY natural tRNA set stock solution and 0.5 µL of the IVT tRNA mix (100 µM each, f.c. 10 µM). For the optimization of the hybrid tRNA set (Figure S6), in addition to the above tRNAs, 1.0 µL of the IVT tRNA stock solution (Group A, B, C, or D containing 50 µM of each tRNA, f.c. 10 µM each) was added. For the experiment in Fig. 4, 0.5 µL of 100 µM of IVT tRNA (f.c. 10 µM) from Group A was added. For the experiment translating sfGFP (Fig. 5, 6, S7, S8, and S9), 0.5 µL of 100 µM (f.c. 10 µM) IVT tRNA^Phe was added. The translation mixture also contained 0.5 mM of each amino acid (refer to figure legend for the Asp concentration) and 2 µM of mRNA. The translation reaction was performed at 37°C for 3 h.

The translation mixture (5 µL) containing 1.5 mg/mL of natural tRNA extract was prepared by adding 0.75 µL of the 10 mg/mL intact natural tRNA extract. The translation mixture for the IVT-SL tRNA set was prepared according to the procedure described in our previous report⁴.

Analysis of translated products

The synthesized model peptides labeled with [¹⁴C]-Asp in the translation reaction mixture were analyzed using 15% polyacrylamide tricine SDS-PAGE followed by autoradiography (Pharos Fx imager, Bio-Rad). Alternatively, reactions with Asp instead of [¹⁴C]-Asp were performed, and the resultant products were analyzed by MALDI-TOF-MS (autoflex maX, Bruker) according to the procedure in the previous report³⁰.

The synthesized model protein, sfGFP labeled with [¹⁴C]-Asp, in the translation reaction mixture was analyzed by 15% polyacrylamide native PAGE followed by autoradiography (Pharos Fx imager, Bio-Rad) and fluorescence detection (λex = 473 nm, LPB filter; Typhoon FLA 9000, GE Healthcare). For data presented in Fig. 4 and Figure S6, reactions using Asp instead of [¹⁴C]-Asp were performed. The products from these reactions were analyzed using fluorescence spectrometry (FP-8500, JASCO).

Data availability

The data supporting the findings of this manuscript are available from the corresponding author upon reasonable request.

Acknowledgments

This study was financially supported by AMED (grant numbers JP21zf0127004 to H.M.), a Grant-in-Aid for Scientific Research(S) (grant number 23H05456 to H.M.), and a Grant-in-Aid for Scientific Research(C) (grant number 21K05270 to T.F.) from the Japan Society for the Promotion of Science and a donation from H. Murakami.

Author contributions

Experiments were designed by T.F. and H.M. Samples were prepared by T.F., R.S., and H.M. Data were acquired by R.S. Figure design and manuscript writing were performed by T.F. and H.M. The project was supervised by H.M.

Competing interests

No potential conflicts of interest were disclosed.

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Ser/Leu-swapped cell-free translation system constructed with natural/in vitro transcribed-hybrid tRNA set

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Abstract

Figures

Introduction

Result and discussion

Synthesis of peptides in a cell-free translation system with the hybrid-Std tRNA set

Accuracy of Ser/Leu-swapping in a cell-free translation system with the hybrid-SL tRNA set

Optimization of the hybrid tRNA set for translating a protein

Ser/Leu-swapped synthesis of a model protein using the hybrid-SL tRNA set

Conclusion

Material and methods

Materials

Preparation of sfGFP mRNAs

Preparation of ΔSLY natural tRNAs

Preparation of tRNA stock solutions

Cell-free translation

Analysis of translated products

Declarations

References

Supplementary Files

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