Multiple Amino Acid-Excluded Genetic Codes for Protein Engineering

Nov 20, 2013 - A “simplified genetic code”, with only 19 amino acids assigned to the sense codons, was recently developed. In this study, we descr...
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Letter pubs.acs.org/synthbio

Multiple Amino Acid-Excluded Genetic Codes for Protein Engineering Using Multiple Sets of tRNA Variants Kazuaki Amikura,†,‡ Yoko Sakai,† Shun Asami,† and Daisuke Kiga*,†,‡ †

Department of Computational Intelligence and Systems Science, Interdisciplinary Graduate School of Science and Engineering, Tokyo Institute of Technology, Midori-ku, Yokohama-shi, Kanagawa 226-8503, Japan ‡ Earth-Life Science Institute, Tokyo Institute of Technology, Meguro-ku, Tokyo 152-8551, Japan S Supporting Information *

ABSTRACT: A “simplified genetic code”, with only 19 amino acids assigned to the sense codons, was recently developed. In this study, we describe novel simplified codes in which multiple amino acids are simultaneously excluded from the universal code. In the simplest code, tryptophan, cysteine, tyrosine, and asparagine codons are assigned to serine by using four kinds of tRNASer variants. The results revealed that various sets of amino acids can easily be excluded from the universal code, using our strategy for genetic code simplification. A simplified genetic code is useful as an engineering tool for the improvement of industrial enzymes and pharmaceuticals, and also provides new insights into the assessment of protein evolution. Simplified codes in which multiple amino acids are simultaneously excluded from the code can be more effective tools than codes excluding only one amino acid. KEYWORDS: amino acid, genetic code, tRNA, protein engineering, evolution

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he 20 canonical amino acids are assigned to the sense codons in the universal code. The assignments are achieved by the functional interactions of aminoacyl-tRNA synthetases (aaRSs), tRNAs, the 20 amino acids, and other components of the translational apparatus. Each amino acid is connected to its cognate tRNA(s) by a distinct aaRS. The 20 groups of aaRS/tRNA pairs assign the 20 canonical amino acids to their sense codons in the universal code. The number of amino acids in the universal code has been increased by the addition of a new aaRS/tRNA pair, by either engineering1,2 or natural emergence.3 In contrast to the success of expanded codes utilizing over 20 amino acids in the sense codons, the restriction of the kinds of amino acids assigned to the sense codons in a code has barely been addressed. However, previous studies have suggested that the number of amino acids has increased since the emergence of the primordial genetic code.4−6 Recently, the construction of a simplified genetic code containing only 19 amino acids was reported, along with the proposal of the artificial evolution of a simplified protein, composed of fewer than 20 amino acids.7 In the first step of simplified genetic code creation with 19 amino acids, we produced unassigned codons by removing an amino acid from the Escherichia coli S30 cell-free translation mixture8 and adding a potent inhibitor of the aminoacyl-tRNA synthetase for the amino acid (Figure 1A). We then reassigned the unassigned codons to Alanine (Ala) or Serine (Ser), by adding a tRNA variant with an anticodon corresponding to the unassigned codons (Figure 1B). As previously proposed,7 the simplified © XXXX American Chemical Society

Figure 1. Schematic view of the simplified genetic code. (A) Diagram of the simplified genetic code without Tyr. The absence of Tyr and the presence of a TyrRS-specific inhibitor result in the lack of Tyr-tRNATyr in the translation mixture. By the addition of a tRNAAla variant with the anticodon loop corresponding to the UAC codon, the UAC codon on mRNA is translated into Ala. The tRNAAla variant is aminoacylated with Ala. (B) The nucleotide sequences of the anticodon stem loop of the tRNAAla variant and the UAC codon on the mRNA. The anticodon loop of tRNAAla was substituted for that of tRNATyr. Positions 32 and 38 in the anticodon loop are numbered.

code will augment the utility of such proteins for clinical9 or industrial use, and thus the exclusion of a specific kind of amino acid is effective for the improvement.9−12 Using the universal code, the creation of a simplified protein by selection from a Special Issue: SB6.0 Received: September 20, 2013

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Figure 2. Eleven newly simplified genetic codes, in which 19 amino acids are assigned to the sense codons. Autoradiograms of [14C]-Leu labeled proteins are shown. The proteins were translated under the conditions noted at the top of each lane. The concentration of each added tRNA and a.a.-SA is provided below. (A) Trp-lacking simplified code using tRNAAla variants: AW_CCA_tri, AW_CCA_loop. In lanes 3−9, 5 μM Trp-SA was used. In lanes 4−6, 0.3 μM, 1 μM, and 4 μM AW_CCA_tri were used. In lanes 7−9, 0.1 μM, 0.3 μM, and 1 μM AW_CCA_loop were used. (B) Cys-lacking simplified code using tRNAAla variants: AC_GCA_tri, AC_GCA_loop. In lanes 3−9, 2 μM Cys-SA was used. In lanes 4−6, 0.02 μM, 0.2 μM, and 2 μM AC_GCA_tri were used. In lanes 7−9, 0.02 μM, 0.2 μM, and 2 μM AC_GCA_loop were used. (C) Tyr-lacking simplified code using tRNAAla variants: AY_GUA_tri, AY_GUA_loop. In lanes 3−9, 1 μM Tyr-SA was used. In lanes 4−6, 0.03 μM, 0.3 μM, and 3 μM AY_GUA_tri were used. In lanes 7−9, 0.03 μM, 0.3 μM, and 3 μM AY_GUA_loop were used. (D) Asn-lacking simplified code using tRNAAla variants: AN_GUU_tri, AN_GUU_loop. In lanes 3−9, 5 μM Asn-SA was used. In lanes 4−6, 0.3 μM, 1 μM, and 3 μM AN_GUU_tri were used. In lanes 7−9, 0.3 μM, 1 μM, and 3 μM AN_GUU_loop were used. (E) Trp-lacking simplified code using a tRNASer variant: SW_CCA_loop. In lanes 3−9, 5 μM Trp-SA was used. In lanes 4−6, 0.3 μM, 1 μM, and 4 μM SW_CCA_loop were used. (F) Cys-lacking simplified code using tRNASer variants: SC_GCA_tri, SC_GCA_loop. In lanes 3−9, 5 μM Cys-SA was used. In lanes 4−6, 0.03 μM, 0.3 μM, and 3 μM SC_GCA_tri were used. In lanes 7−9, 0.03 μM, 0.3 μM, and 3 μM SC_GCA_loop were used. (G) Tyr-lacking simplified code using a tRNASer variant: SY_GUA_loop. In lanes 3−9, 1 μM Tyr-SA was used. In lanes 4−6, 0.03 μM, 0.3 μM, and 3 μM SY_GUA_loop were used. (H) Asn-lacking simplified code using a tRNASer variant: SN_GUU_loop. In lanes 3−9, 5 μM Asn-SA was used. In lanes 4−6, 0.3 μM, 1 μM, and 3 μM SN_GUU_loop were used.

random mutagenesis library has been hampered by the reappearance of codons for the amino acid to be removed from the library.9 By contrast, the use of a simplified genetic code ensures that every protein sequence translated from any mRNA in the library does not include the amino acid(s) to be eliminated, through selection cycles. In this study, we describe the exclusion of multiple amino acids from the universal code, by using multiple sets of tRNA variants. Our results confirmed the general applicability of our method to simplify the code. In the future, we will easily create even simpler genetic codes, as effective tools for the improvement of enzymatic functions or the assessment of early stages of protein evolution. In each modified genetic code lacking one amino acid, we first produced the unassigned codon(s) corresponding to tryptophan (Trp), cysteine (Cys), tyrosine (Tyr), or asparagine (Asn) in the universal code. The unassigned codons were generated by removing the amino acid from the cell-free translation mixture and adding the specific a.a.-SA, which is a potent inhibitor of the aminoacyl-tRNA synthetase corresponding to the amino acid. The production of the unassigned codon was confirmed by the absence of protein synthesis (Figure 2A-

H, Lane 3). As a result, we produced the unassigned codon(s) for each of the four amino acids in the universal code. In all lanes without an amino acid, no premature polypeptides produced by ribosome stalling at the unassigned codon were detected. We speculate that the premature polypeptides were not stably folded and were rapidly degraded by proteases in the E. coli extracts.8 Different concentrations of the added a.a.-SA were required to prepare the unassigned codons, due to various factors. First, a few amino acids are known to be produced in the metabolism of S30 cell-free extracts,13 even after the complete removal of the amino acid. Second, the various a.a.-SAs have distinct inhibition constants.14,15 In addition, the proportion of each amino acid removed from the translation mixture by dialysis might be different. For instance, a large amount of Asn is retained, as compared to the removal of Trp, Cys, and Tyr. When the amino acid is only partially removed, and a significant quantity remains in the translation mixture, then more a.a.-SA corresponding to the amino acid is needed. To reduce the number of amino acids assigned to the sense codons in the universal code, we prepared tRNAAla,UGC variants and tRNASer,UGA variants. As in our previous work, the B

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the Tyr-lacking and Asn-lacking codes, the tRNA variants read the codon involving the G·U wobble pair (Supplementary Table S1). By using sets of multiple tRNASer,UGA variants and a.a.-SAs, we created new, simplified genetic codes, in which up to four amino acids are excluded (Figure 3). The simplified code

anticodon loops of these tRNAs were substituted with the anticodon loop of the wild-type E. coli K-12 tRNA corresponding to the unassigned codon. For example, when we constructed the Tyr-lacking simplified code, in which Ala is assigned to Tyr codons, we used the tRNA variant with the anticodon loop of wild-type E. coli K-12 tRNATyr,GUA, with reference to the tRNA database (Figure 1b).16 We also prepared tRNA variants in which only the anticodon triplet sequence was substituted. We named each tRNA variant according to certain rules (Table 1). Table 1. tRNA Variants Used for the Construction of the Simplified Genetic Code namea

sequenceb

AW_CCA_tri AW_CCA_loop AC_GCA_tri AC_GCA_loop AY_GUA_tri AY_GUA_loop AN_GUU_tri AN_GUU_loop SW_CCA_loop SC_GCA_tri SC_GCA_loop SY_GUA_loop SN_GUU_loop

UUCCAAC CUCCAAA UUGCAAC UUGCAAA UUGUAAC CUGUAAA UUGUUAC CUGUUAA CUCCAAA CUGCAAA UUGCAAA CUGUAAA CUGUUAA

The name was chosen according to the following rules. The first letter, “A” and “S”, means that the variant is a derivative of E. coli K-12 tRNASer,UGA and tRNAAla,UGC, respectively. The second letter, “C”, refers to the 1-letter abbreviation of the amino acid corresponding to the anticodon of the tRNA variant. The three letters in between the underscores show the anticodon of the tRNA variant. The word after the second underscore defines the region of the substituted sequence, and “loop” and “tri” represent the anticodon loop and the anticodon triplet, respectively. For example, when the anticodon loop of tRNASer,UGA was substituted for the wild-type tRNACys,GCA, the tRNASer,UGA variant was named “SC_GCA_loop”. bThe sequence of the anticodon loop of the tRNA variant. a

Figure 3. Novel simplified genetic codes in which multiple amino acids are simultaneously excluded from the universal code. (A) Simplified 18 amino acid code using a tRNASer variant: the SW_CCA_loop, and the SC_GCA_loop. In lanes 3−4, the concentrations of both Trp-SA and Cys-SA were 5 μM. In lane 4, 1 μM the SW_CCA_loop and 0.4 μM the SC_GCA_loop were used. (B) Simplified 17 amino acid code using a tRNASer variant: the SW_CCA_loop, the SC_GCA_loop, and the SY_GUA_loop. In lanes 3−4, Trp-SA, Cys-SA, and Tyr-SA were used at 5 μM. In lane 4, 1 μM the SW_CCA_loop, 0.4 μM the SC_GCA_loop, and 1 μM the SY_GUA_loop were used. (C) Simplified 16 amino acid code using a tRNASer variant: the SW_CCA_loop, the SC_GCA_loop, the SY_GUA_loop, and the SN_GUU_loop. In lanes 3−4, Trp-SA, Cys-SA, Tyr-SA, and Asn-SA were used at 5 μM. In lane 4, 1 μM SW_CCA_loop, 0.4 μM SC_GCA_loop, 1 μM SY_GUA_loop, and 0.4 μM SN_GUU_loop were used.

We constructed 11 newly simplified genetic codes using one tRNA variant (Figure 2). We previously developed a Trplacking code, using the AW_CCA_loop, and a Cys-lacking code, using the SC_GCA_loop.7 As with the tRNA variants used in the previous study (Figure 2A lanes 7−9 and Figure 2F lanes 7−9), the SW_CCA_loop and the AC_GCA_loop allowed the construction of other Trp-lacking (Figure 2E lanes 4−6) and Cys-lacking codes (Figure 2B lanes 7−9), respectively. In addition, we newly constructed Tyr-lacking codes (Figure 2C lanes 4−9, Figure 2G lanes 4−6) and Asnlacking codes (Figure 2D lanes 4−9, Figure 2H lanes 4−6). We considered the codons that became unassigned by the addition of the aaRS inhibitor to be reassigned to Ala or Ser, because the protein production increased in a manner dependent upon the concentration of the tRNA variant, in most cases. We also confirmed that the efficiency of protein synthesis by the simplified code is comparable to that of the universal code (Figure 2A lanes 1, 6, and 9; 2B lanes 1, 6, and 9; 2C lanes 1, 6, and 9; 2D lanes 1, 6, and 9; 2E lanes 1 and 6; 2F lanes 1, 6, and 9; 2G lanes 1 and 6; 2H lanes 1 and 6). When we constructed the Cys-lacking codes, the tRNA variants with the GCA anticodon read the Cys codon UGU involving the G·U wobble pair (Supplementary Table S1). Likewise, when we constructed

excluding both Trp and Cys from the universal code was constructed by the SW_CCA_loop and SC_GCA_loop sets, Trp-SA, and Cys-SA (Figure 3A, lane 4). In order to further remove Tyr from the 18 amino acid simplified code, we used the SY_GUA_loop and Tyr-SA (Figure 3B, lane 4). Finally, we constructed the most simplified code, in which only 16 amino acids are assigned to sense codons, by using the SW_CCA_loop, SC_GCA_loop, SY_GUA_loop, and SN_GUU_loop sets, Trp-SA, Cys-SA, Tyr-SA, and Asn-SA (Figure 3C, lane 4). We confirmed that the efficiency of protein synthesis by each simplified genetic code was comparable to that of the universal C

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Preparation of tRNA Transcripts. The tRNA variants were prepared by runoff transcription, using T7 RNA polymerase and a PCR-amplified linear DNA template. The variants were purified by 8% polyacrylamide gel (29:1) electrophoresis, in a gel prepared with 8 M urea and TBE. After the denaturing PAGE, the band was located by UV shadowing, excised, and eluted by rotation for 12 h in 1 mL of 0.3 M NaCl. Finally, the eluted product was filtered by a MillexHV Syringe Filter Unit, 0.45 μM pore size (Merck Millipore, Massachusetts, USA). Cell-Free Protein Expression. The E. coli S30 cell-free protein synthesis method was used in this study. The composition of the cell-free protein synthesis reaction was the same as that previously described,8 except for the omission of a specific amino acid, and the addition of the tRNA variant and the a.a.-SA (5′-O-[N-(L-a.a.)sulfamoyl]adenosine aminoacyl adenylate analogues, Integrated DNA Technologies, Coralville, IA). The S30 extract was prepared from the E. coli BL21 DE3 strain. The batch mode was employed, with 20-μL reaction volumes and a reaction time of 1 h. Detection of Radiolabeled Products. Translation of CAT, Ras, and StAv was performed using the 20 μL scale batch mode of synthesis at 37 °C for 1 h with the components described above, except for the addition of [14C]-Leu. The products were directly analyzed on 12% Bis−Tris gels with MES running buffer (50 mM MES, 50 mM Tris−base, 3.47 mM SDS, 1.0 mM EDTA, pH 7.3). Scanning was performed using an image analyzer, FLA-5000 (FUJI), and an imaging plate, BAS-IP MS 2040 (FUJI), to measure the radioactivity of the products.

code. The results suggested that over four amino acids can be excluded simultaneously, by using several a.a.-SA sets and tRNA variants. In this study, we also constructed a simplified code by using tRNA variants with an anticodon triplet substitution, rather than an anticodon loop substitution. Previous studies showed that changing the highly conserved base in the anticodon loop perturbed the efficiency or fidelity of translation.17,18 In our study, however, the efficiency of protein synthesis was similar between the two types of tRNA variants with the same anticodon (Figure 2A lanes 5 and 9, 2B lanes 6 and 9, 2C lanes 6 and 9, 2D lanes 6 and 9, and 2F lanes 6 and 9). However, we could not determine whether the coding fidelity of the protein synthesis is the same between the two cases. Further experiments, such as amino acid composition analyses as in the previous study,7 will determine the coding fidelity. To obtain a simplified protein by directed evolution, the simplified code lacking multiple amino acids will be a more effective tool than codes excluding one amino acid. In general, using the universal code, the construction of a simplified protein by selection from a random mutagenesis library has been hampered by the reappearance of codons for the amino acid that should be removed from the library.7 When removing multiple amino acids from a protein, the reappearance probability becomes higher. With the use of a simplified code, proteins without the amino acids to be eliminated are produced from any mRNA in the library. Both Ala and Ser, which are used in scanning mutagenesis,19,20 are considered as having an equal or higher advantage for protein folding or solubility, as compared with the other amino acids in the universal code. Ala, which appears in the library, does not interfere with protein structures, as compared to the other amino acids.21 Correct protein folding in the presence of many alanine substitutions has been demonstrated.22,23 In the library, Ser appears to contribute more favorably to protein solubility than any of the other amino acids in the universal code.24 In addition to the potential advantage of the translational products with Ala and/or Ser substitutions, the versatile compositions of the tRNAAla and tRNASer variants that we have demonstrated in this work will be crucial for the construction of even simpler codes encoding fewer than 16 amino acids. Such simplified codes including only the amino acids that were considered to be used in putative primitive codes5,6,25 will facilitate the assessment of the early stages of protein evolution, by providing a large number of artificially evolved, simplified proteins.

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ASSOCIATED CONTENT

S Supporting Information *

Supporting Table S1. This information is available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Author

*Tel & Fax: +81-45-924-5213. E-mail: [email protected]. Author Contributions

K.A. and D.K. conceived the study. K.A. and D.K. designed the experiments. K.A., Y.S., and S.A. performed the experiments. All of the authors participated in the interpretation of the results and the preparation of the manuscript.

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Notes

The authors declare no competing financial interest.

METHODS DNA Constructs. For the expression of His-tagged ras, we used the pK7 plasmid.26 The expression plasmid for chloramphenicol acetyltransferase with an N-terminal His tag was constructed previously.7 The green fluorescent protein (GFP) genes were cloned into the pGFP plasmid.27 StAv was synthesized from an oligonucleotide with a T7 promoter. Genes encoding tRNA variants were cloned into the pUC119 plasmid (Takara, Shiga, Japan). Each tRNA variant was created by site-directed mutagenesis from a plasmid encoding tRNAAla,UGC and tRNASer,UGA.7 In the mutagenesis experiments, polymerase chain reaction (PCR) amplification was performed with KOD-plus (Toyobo, Tokyo, Japan), using the following PCR conditions: 2 min at 94 °C for 1 cycle, followed by 20 cycles of 0.25 min at 94 °C, 0.5 min at 65 °C and 3.2 min at 68 °C.

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ACKNOWLEDGMENTS We thank Takanori Kigawa and Shigeyuki Yokoyama for providing the Ras constructs. The authors thank Akio Kawahara-Kobayashi and Masahiko Uchiyama for providing advice and helpful discussions. The authors are grateful for Grants-in-Aid from KAKENHI programs [19680016, 21680026, 23119005, and 23650155 to D.K.]; Japan Society for the Promotion of Science (JSPS); Ministry of Education, Culture, Sports, Science and Technology (MEXT); and the Industrial Technology Research Grant Program in 2005, from the New Energy and Industrial Technology Development Organization (NEDO) (to D.K.). Funding for the open access charge was provided by the KAKENHI program [23119005 to D.K.]. D

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