ATP Recycling with Cell Lysate for Enzyme-Catalyzed Chemical

Nov 9, 2016 - E. coli lysate efficiently catalyzes acetyl phosphate-driven ATP regeneration in several important biotechnological applications. The ut...
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ATP Recycling with Cell Lysate for Enzyme-Catalyzed Chemical Synthesis, Protein Expression and PCR Apostolos Alissandratos,* Karine Caron, Thomas D. Loan, James E. Hennessy, and Christopher J. Easton* Research School of Chemistry, Australian National University, Canberra ACT 2601, Australia ABSTRACT: E. coli lysate efficiently catalyzes acetyl phosphatedriven ATP regeneration in several important biotechnological applications. The utility of this ATP recycling strategy in enzymecatalyzed chemical synthesis is illustrated through the conversion of uridine to UMP by the lysate from recombinant overexpression of uridine kinase with the E. coli. The UMP is further transformed into UTP through sequential phosphorylations by kinases naturally present in the lysate, in high yield. Cytidine and 5-fluorouridine also give the corresponding NMPs and NTPs with this system. Cell-free protein expression with a processed extract of lysate also proceeds readily when, instead of adding the required NTPs, all four are produced in situ from the NMPs, using acetyl phosphate and relying on endogenous kinase activity. Similarly, dNMPs can be used to produce the dNTPs necessary for DNA synthesis in PCR. These cheap alternative protocols showcase the potential of acetyl phosphate and ATP recycling with readily available cell lysate.

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of the auxiliary enzyme, by instead making use of the lysate that is normally regarded as a waste byproduct of recombinant protein production. Related strategies have recently been reported for NADH- and NADPH-dependent recombinant enzymes, recycling these cofactors using glucose and cell extract,8−10 but for ATP regeneration with acetyl phosphate, this has previously only been evaluated by Kim and coworkers.11,12 They investigated the use of an ATP-dependent reaction catalyzed by a recombinant enzyme coupled to endogenous acetate kinase-catalyzed ATP recycling, for the production of sugar nucleotides. Although their initial findings were positive, 11 they subsequently concluded that the endogenous acetate kinase activity was insufficient, and instead it was necessary to also overexpress this enzyme.12 In contrast, here we show that expression of a recombinant uridine kinase mutant from Thermus thermophilus,13 in the commonly employed E. coli strain BL21(DE3), produces a lysate with sufficient acetate kinase activity to support not only efficient conversion of uridine to UMP, but also further sequential phosphorylations of the UMP to produce UTP, in high yield. The conversion of UMP to UTP is catalyzed by NMP, NDP and acetate kinases naturally present in the lysate. This cheap and easy to prepare biocatalyst mixture also transforms cytidine and the unnatural nucleoside 5-fluorouridine into the corresponding NMPs and NTPs, demonstrating the utility of the recombinant enzyme in combination with acetyl phosphate, ATP recycling, and endogenous kinases in cell lysate for NTP

odern synthetic and biological chemistry relies increasingly on the use of purified enzymes to carry out challenging reactions cleanly and selectively, under mild conditions.1−3 However, a major constraint on the more widespread use of enzymes is their dependence on expensive cofactors, particularly ATP.4 The requirement for stoichiometric amounts of ATP has been addressed through recycling, by incorporation of an auxiliary enzyme-catalyzed step.4,5 Most relevant to this study, Whitesides and co-workers5,6 have driven ATP regeneration from ADP catalyzed by acetate kinase, using acetyl phosphate as the phosphate donor (Figure 1). Acetyl

Figure 1. Regeneration of ATP from ADP using acetyl phosphate.

phosphate is economical and easy to synthesize for large-scale use. Nevertheless, this method of recycling necessitates the use of an additional enzyme, contributing significantly to process cost.7 Due to limitations of this type, ATP-dependent enzyme technologies remain relatively unexploited. To address this, we investigated the possibility of utilizing the cell lysate from the overexpression of an ATP-dependent enzyme as the catalyst for not only the ATP-dependent reaction but also the auxiliary recycling reaction. Our aim was to avoid the requirement for separate production and isolation © XXXX American Chemical Society

Received: September 23, 2016 Accepted: November 9, 2016 Published: November 9, 2016 A

DOI: 10.1021/acschembio.6b00838 ACS Chem. Biol. XXXX, XXX, XXX−XXX

Letters

ACS Chemical Biology

Figure 2. ATP recycling by E. coli lysate to drive cell-free biotechnological processes.

production, as an alternative to current commercial processes.14,15 More generally, we observed that in addition to UTP, CTP, and 5-fluoro-UTP, the lysate catalyzes production of ATP, GTP, dATP, dCTP, dGTP, and dTTP from the respective NMPs and dNMPs. We therefore became interested in the combination of this synthetic process with applications where NTPs and dNTPs are building blocks for nucleic acid synthesis. Acetyl phosphate has already been used with cell-free protein expression for ATP16,17 and GTP recycling,16 but not to substitute NMPs for all four NTPs required for mRNA synthesis. Calhoun and Swartz18 replaced NTPs with NMPs in a cell-free transcription-translation protocol, driven by glucose with additional compounds added to support glycolysis, and based on bacteria grown in glucose supplemented rich medium.19,20 Elaborations to this glycolysis-driven system have also been reported.21,22 Here, we report a complementary procedure, using acetyl phosphate and an extract of the lysate of E. coli BL21 Star (DE3), for mRNA synthesis by T7 RNA polymerase during protein expression using NMPs. This strain of E. coli is identical to BL21(DE3) except for the absence of RNase E, thus allowing mRNA a longer half-life in the cell-free reaction. Employing glucose in this system did not result in either NTP production or protein synthesis, and in fact added ATP was converted back to AMP. In addition, we describe utilizing the dNTPs produced by the cell lysate from the cheaper and more stable dNMPs for production of DNA through addition of the lysate mixture to a polymerase chain reaction (PCR). Together these applications greatly extend the utility of cell-free methodologies for the synthesis and processing of nucleotides in various important biomolecular processes that govern the flow of genetic information (Figure 2). Initially, we probed E. coli cell lysate for ADP to ATPtransforming enzymes. Adding acetyl phosphate resulted in a very high turnover, establishing the presence of a high concentration of active acetate kinase (800 units mL−1 lysate, determined at 37 °C and pH 7.5), the enzyme known to catalyze this reaction (Figure 1). The glycolytic intermediates fructose-1,6-bisphosphate and 3-phosphoglycerate afforded much less ATP production, while glucose-6-phosphate and glucose caused ATP degradation to AMP. We then investigated ATP recycling with a mutant (Tyr93His) uridine kinase. The gene encoding this enzyme in Thermus thermophilus was cloned into the pETMCSIII expression vector23 for production of protein with an N-(His)6-tag, to facilitate purification. Isolated protein that had been expressed in E. coli BL21(DE3) displayed the expected activity with uridine and cytidine,13 as well as for the unnatural nucleoside 5-fluorouridine, showing that the enzyme catalyzes production of nucleotides and analogues of importance as chemotherapeutic agents.24,25

The crude lysate from recombinant expression of the uridine kinase also catalyzed the phosphorylation of uridine to UMP, and the conversion required only substoichiometric amounts of ATP when acetyl phosphate was added. This uridine kinase activity must be due to the overexpressed protein, as a control experiment showed that it is lacking in untransformed E. coli lysate. As expected with a crude enzyme mixture, some method optimization was required to obtain good product yields. Initially, some of the uridine was consumed in a side reaction, but once this had been identified as uracil formation reversibly catalyzed by endogenous uridine phosphorylase, it was effectively blocked through the addition of uracil. It follows that the lysate is capable of producing UMP from either uracil or uridine, but uracil requires ribose-1-phosphate as a cosubstrate so it provides no practical or economic advantage. For reactions carried out with less than 1% cell lysate in TrisHCl buffer, after reaction times of less than an hour, UMP was the major product (>75%), with no detectable UDP and only small amounts of UTP (∼5%) being formed. With 5% lysate and a large excess of acetyl phosphate over uridine, the major product was UTP, so there are naturally present NMP, NDP, and acetate kinases that work in concert with the expressed uridine kinase, to catalyze the conversions of uridine to UMP, then UDP followed by UTP, using acetyl phosphate. With 5% lysate but only a 1.5-fold excess of acetyl phosphate, UMP is the major product. These results show that the activity of the expressed uridine kinase is greater than that of the NMP kinase, and the conditions can therefore be manipulated to produce mainly either UMP or UTP. Given the presence of the NMP, NDP, and acetate kinases in the lysate, we were also able to use AMP in place of the more expensive ATP in these experiments. Various AMP concentrations of 0.2 mM and above resulted in similar reaction rates, but they were lower at 0.1 mM. No reaction occurred without adding AMP, demonstrating the crucial role of this species in the overall catalysis. The background levels of AMP, ADP, and ATP in the cell lysate total less than 10 μM. High concentrations of lysate, particularly above ∼20%, reduced the product yield based on acetyl phosphate. This is probably a result of hydrolysis catalyzed by phosphatases. The impact of this effect was reduced through the use of phosphate buffer in place of Tris-HCl, presumably due to phosphate inhibition of the hydrolytic enzymes. The concentration of phosphate had no apparent effect on the rates and extents of conversion of uridine to UMP and UTP when tested at concentrations ranging from 0.1 to 0.4 M, or on the initial reactions at 0.05 M, but this lowest concentration was insufficient to buffer acetate production from acetyl phosphate and maintain extended reaction. In phosphate, the use of excess acetyl phosphate resulted in rapid and complete production of UTP from uridine. Alternatively, using less acetyl phosphate resulted in B

DOI: 10.1021/acschembio.6b00838 ACS Chem. Biol. XXXX, XXX, XXX−XXX

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of such a biological system for NTP synthesis is its compatibility with many of the biotechnological applications where these compounds are required, as described above for the production and use of recombinant uridine kinase. In protein synthesis, mRNA is produced from NTPs during transcription and prior to translation. Processed cell lysates are utilized for cell-free protein expression, and a normal protocol is to add NTPs, as well as exogenous creatine kinase and creatine phosphate to maintain NTP levels and sustain protein expression.26−28 We studied the possibility of instead utilizing the S30 cell lysate of E. coli BL21 Star (DE3) for in situ generation of the required NTPs from NMPs, driven by acetyl phosphate-endogenous acetate kinase, without adding creatine kinase or creatine phosphate. In that event, high levels of the test protein, N-(His)6-peptidyl-prolyl cis−trans isomerase B (N(His)6-PpiB), were obtained through in vitro translation (Figure 4a, lane 2), similar to those produced using the parent protocol (lane 1). By contrast, the use of NMPs and glucose did not result in NTP or protein synthesis (lane 3), probably because lysate capable of generating ATP from glucose is likely to require growth of the cells on a glucose-rich medium. Omitting acetyl phosphate also resulted in no protein expression (lane 4).

slower and less extensive phosphorylation and the production of mainly UMP, but high yields based on acetyl phosphate. An additional advantage with this buffer is that acetyl phosphate could be prepared through addition of acetic anhydride to phosphate solution for direct use. Illustrative results obtained (over three replicates) using the protocol developed in this way and described in the Methods section are shown in Figure 3. In this representative example,

Figure 3. Conversion of uridine (2 mM) to UTP catalyzed by lysate of E. coli BL21(DE3) from overexpression of uridine kinase, with AMP (0.2 mM) and acetyl phosphate (60 mM).

added AMP is converted to ATP within minutes, which then remains at a similar concentration for the duration of the experiment, demonstrating the ample sufficiency and stability of the recycling system. After 4−5 h, near quantitative conversion of uridine to UTP is observed (95−100%). This corresponds to a turnover number of 20−30 for ATP regeneration depending on the extent to which the UDP to UTP conversion is catalyzed by ATP-dependent NDP kinases or acetyl phosphate using acetate kinase. The continued production of UTP throughout the course of this experiment, until almost all of the uridine is converted, shows the robustness of the catalytic system. Under these conditions, acetyl phosphate is in excess but when used as the limiting reagent gives mainly UMP with a trace of UTP in a combined yield of 70% based on the acetyl phosphate. The same approach was applied for the production of CMP and CTP from cytidine as well as 5-fluoro-UMP and 5-fluoroUTP from 5-fluorouridine. A small amount of 5-fluorouridine was lost to 5-fluorouracil and of cytidine to cytosine and uridine, the latter reaction being catalyzed by cytidine deaminase, but even so CTP and 5-fluoro-UTP were each produced in good yield without addressing these side reactions. The corresponding NDP was not detected in either case. There was no apparent change to the efficiency of the ATP recycling in all these reactions. ATP or AMP could be used, which in the latter case was rapidly converted to ATP, and in all experiments the ATP was maintained by the acetyl phosphate-driven endogenous acetate kinase activity. The efficiency of the synthesis of UTP, CTP, and 5-fluoroUTP prompted us to investigate the generality of NTP production with ATP recycling. Accordingly, ATP, GTP, dATP, dCTP, dGTP, and dTTP were all prepared from the respective NMPs and dNMPs. High yields were obtained in each case, but ATP and dATP formed fastest. The catalytic activity of all the NMP and NDP kinases required to catalyze these ATP-dependent phosphorylations in E. coli must therefore be retained in the lysate. One of the major advantages

Figure 4. (a) SDS-PAGE analysis of purified N-(His)6-PpiB (19.2 kDa) obtained using NTPs and creatine phosphate−creatine kinase (lane 1), NMPs and acetyl phosphate (lane 2), NMPs and glucose (lane 3), and NMPs but no acetyl phosphate (lane 4) [(M) is NEB 2− 212 kDa marker]. (b) Agarose gel analysis of PCR products obtained using dNMP phosphorylation mixture (lane 1), dNMP mixture but no lysate (lane 2), and lysate but no dNMP mixture (lane 3) as source of dNTPs [(L) is NEB 1 kb DNA ladder]. Arrows indicate expected product positions. (c) Conversion of the mixture of dNMPs to the dNTPs used in the PCR experiment described in the Methods. C

DOI: 10.1021/acschembio.6b00838 ACS Chem. Biol. XXXX, XXX, XXX−XXX

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tetrabutylammonium phosphate 5 mM and tetrabutylammonium phosphate 5 mM in methanol, monitoring at 259 nm. Construction of pETMCSIII-Udk. The DNA sequence for the uridine kinase gene from Thermus thermophilus HB8 (Uniprot Q5SKR5, with Tyr93His) was commercially synthesized codon optimized for E. coli (GeneArt, Germany). The gene was then subcloned into expression vector pETMCSIII, for protein expression with an N-terminal (His)6-tag, under the control of a T7 promoter. Integrity of the coding region was confirmed by sequencing (ABRF, John Curtin School of Medical Research). Preparation of E. coli Lysate with Overexpressed Uridine Kinase. Luria−Bertani medium (1 L) supplemented with 50 mg of ampicillin was inoculated with a 10 mL starter culture of E. coli BL21(DE3) transformed with pETMCSIII-Udk. Cells were grown for 18 h (37 °C, 180 rpm), then harvested by centrifugation (4000g, 15 min). The cell pellet was resuspended in 5 mL of Tris-acetate buffer (10 mM, pH 8.3) supplemented with 16 mM KOAc, 14 mM MgOAc, 1 mM phenylmethylsulfonylfluoride, and 1 mM dithiothreitol. Lysis was performed in a French pressure cell followed by high-speed centrifugation (20000g, 1 h) for insoluble debris removal. The supernatant, which contained 0.45 units mL−1 of uridine kinase activity (37 °C, pH 7.4), was frozen on dry ice and stored at −80 °C for subsequent use. Synthesis of Aqueous Acetyl Phosphate Solution. Acetyl phosphate was prepared as a ∼1 M aqueous solution from acetic anhydride and phosphoric acid as previously described6 and used directly in subsequent reactions. Synthesis of UTP from Uridine. Synthesis of UTP was carried out in sodium phosphate buffer (0.2 M, pH 7.4) containing uridine (2 mM), AMP (0.2 mM), uracil (6 mM), acetyl phosphate (60 mM), MgCl2 (10 mM), and KCl (100 mM). E. coli lysate from overexpression of uridine kinase (44 μL per mL of reaction volume) was added as catalyst, and the mixture was incubated at 37 °C. To monitor UTP synthesis, samples were drawn at set intervals and quenched with the addition of an equal volume of 0.2% sodium dodecyl sulfate, then directly analyzed by HPLC. The results of this analysis are illustrated in Figure 3. Synthesis of UMP from Uridine. Synthesis of UMP was carried out as described above for the synthesis of UTP, except that 3 mM acetyl phosphate was used. After 5 h, HPLC analysis showed the production of UMP, at 97% yield based on uridine and 65% based on acetyl phosphate, together with UTP (∼5% based on acetyl phosphate). Cell-Free Protein Synthesis Reactions. S30 cell-free extract for cell-free protein synthesis was prepared from E. coli BL21 Star (DE3), and synthesis of N-(His)6-PpiB was performed using a previously described procedure27,30 with the following modifications where required. NTPs were replaced with equal concentrations of NMPs (0.8 mM for CMP, GMP, UMP; 1.2 mM for AMP). When acetyl phosphate or glucose was added, creatine phosphate was replaced with 80 mM of the respective substrate, and creatine kinase was omitted. Synthesis of dNTPs from dNMPs. For production of dNTPs to be used in PCR, 0.5 mL reactions were performed in HEPES buffer (55 mM, pH 7.5) containing dAMP, dCMP, dGMP, and dTTP (10 mM each), magnesium acetate (15 mM), acetyl phosphate (100 mM), ATP (1 mM), and 25 μL of S30 cell extract. The reactions were incubated at 37 °C for 4 h, and the dNTP concentrations were measured by HPLC (Figure 4c). PCR with Synthesized dNTPs. PCR was performed in 50 μL reactions with 4 ng of template DNA (pETMCSIII-Udk), 0.2 μM each of forward (5′-CGACTCACTATAGGGAGACCACAAC-3′) and reverse (5′-CCTTTCGGGCTTTGTTAGCAG-3′) primer (for the production of 0.8 kb amplicon), 1.25 units of Taq polymerase and 10 μL of the combined dNTP synthesis reaction that had been carried out for 4 h and filtered through an Ultracel-50 centrifugal filter. Control reactions were performed either in the absence of cell lysate or in the absence of dNMPs. Thermal cycle, 96 °C initial denaturation, 4 min; 30 cycles [96 °C denaturation, 15 s; 55 °C annealing, 15 s; 72 °C extension, 30 s], and 72 °C final extension, 5 min.

dNTPs are required for the DNA polymerase-catalyzed synthesis of DNA, commonly used in PCR applications.29 As we had already established the ability of the acetyl phosphateendogenous kinase system to generate each dNTP separately, their combined production for use in PCR was considered. dATP, dCTP, dGTP, and dTTP were all produced within a short time frame from a mixture of the corresponding dNMPs (Figure 4c). Although the amount of dTTP was much lower than those of the others, after brief centrifugation to remove nuclease activity that could degrade DNA, addition of the mixture to a PCR reaction with Thermus aquaticus (Taq) DNA polymerase and a plasmid containing a DNA target sequence (udk from Thermus thermophilus, 0.8 kb) along with suitable primers resulted in amplified DNA (Figure 4b). Control experiments established that the dNMPs added to the lysate were the source of the dNTP building blocks in the PCR experiment. In conclusion, together these results demonstrate the versatility of acetyl phosphate-driven ATP regeneration catalyzed by E. coli lysate and its compatibility with a range of applications. E. coli is already widely employed in biotechnology, and no engineering is required. The lysate is a cheap and easy to prepare biocatalyst, often a waste byproduct. Very little processing of the lysate is necessary, to allow use of the cellular components with limited interference from background activities, without the need for enzyme purification. ATP recycling by acetate kinase activity in the lysate is sufficient to simultaneously support multiple ATP-dependent enzymecatalyzed chemical processes, natural and unnatural nucleotide synthesis by an overexpressed recombinant enzyme and in situ conversion of the NMPs to NTPs catalyzed by naturally present kinases. The synthesis of NTPs and dNTPs driven by acetyl phosphate and endogenous kinases in lysate is compatible with important applications in which they are required. It allows the use of the much cheaper and more stable NMPs and dNMPs for production of mRNA in protein synthesis and as starting materials for PCR. Given the much greater proportional cost of AMP and ATP, and the other NMPs and NTPs, in these applications, the relative expense of the acetyl phosphate is quite minor. Moreover, it can be generated from acetic anhydride (current cost ∼ US$50 per kg from laboratory suppliers) and phosphate solution and then used directly in the applications described above. These factors considered, acetyl phosphate-driven ATP recycling by cell lysate appears to have broad potential in biotechnology.

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METHODS

General Methods. Chemical reagents used including nucleosides, (d)NMPs, (d)NDPs, and (d)NTPs were purchased from SigmaAldrich. Taq DNA polymerase, T7 RNA polymerase, and broad range (2−212 kDa) molecular weight markers were from New England BioLabs (MA, USA). Acetic anhydride and H3PO4 (85%) used for acetyl phosphate production were ACS grade. Plasmid DNA for cellfree protein synthesis was prepared with the Qiagen Plasmid Maxi kit, from E. coli DH5α/pND1098. Agarose DNA gels were run on a Wide Mini-Sub Cell GT system with GelRed nucleic acid gel stain. SDSPAGE gels were run on a Mini-PROTEAN Tetra system and stained with Bio-Safe Coomassie Blue stain from Bio-Rad (CA, USA). Proteins were purified with a His-GraviTrap Kit from GE Healthcare. Ultracell centrifugal devices were purchased from Millipore (MA, USA). HPLC analysis of nucleosides and nucleotides was performed on an Agilent 1100 system with an Alltima HP C18 column (5 μ, 250 × 4.6 mm with a 7.5 × 4.6 mm guard) and gradient elution (87−70%) with pH 5.0 aqueous ammonium dihydrogen phosphate 60 mM/ D

DOI: 10.1021/acschembio.6b00838 ACS Chem. Biol. XXXX, XXX, XXX−XXX

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(17) Kim, D. M., and Swartz, J. R. (1999) Prolonging cell-free protein synthesis with a novel ATP regeneration system. Biotechnol. Bioeng. 66, 180−188. (18) Calhoun, K. A., and Swartz, J. R. (2005) An economical method for cell-free protein synthesis using glucose and nucleoside monophosphates. Biotechnol. Prog. 21, 1146−1153. (19) Calhoun, K. A., and Swartz, J. R. (2005) Energizing cell-free protein synthesis with glucose metabolism. Biotechnol. Bioeng. 90, 606−613. (20) Kim, D. M., and Swartz, J. R. (2001) Regeneration of adenosine triphosphate from glycolytic intermediates for cell-free protein synthesis. Biotechnol. Bioeng. 74, 309−316. (21) Kim, T. W., Kim, H. C., Oh, I. S., and Kim, D. M. (2008) A highly efficient and economical cell-free protein synthesis system using the S12 extract of Escherichia coli. Biotechnol. Bioprocess Eng. 13, 464− 469. (22) Kim, T. W., Keum, J. W., Oh, I. S., Choi, C. Y., Kim, H. C., and Kim, D. M. (2007) An economical and highly productive cell-free protein synthesis system utilizing fructose-1,6-bisphosphate as an energy source. J. Biotechnol. 130, 389−393. (23) Neylon, C., Brown, S. E., Kralicek, A. V., Miles, C. S., Love, C. A., and Dixon, N. E. (2000) Interaction of the Escherichia coli replication terminator protein (Tus) with DNA: A model derived from DNA-binding studies of mutant proteins by surface plasmon resonance. Biochemistry 39, 11989−11999. (24) Van Rompay, A. R., Norda, A., Linden, K., Johansson, M., and Karlsson, A. (2001) Phosphorylation of uridine and cytidine nucleoside analogs by two human uridine-cytidine kinases. Mol. Pharmacol. 59, 1181−1186. (25) Zhang, X. M., Lee, I., and Berdis, A. J. (2005) A potential chemotherapeutic strategy for the selective inhibition of promutagenic DNA synthesis by nonnatural nucleotides. Biochemistry 44, 13111− 13121. (26) Spirin, A. S., Baranov, V. I., Ryabova, L. A., Ovodov, S. Y., and Alakhov, Y. B. (1988) A continuous cell-free translation system capable of producing polypeptides in high yield. Science 242, 1162− 1164. (27) Ozawa, K., Headlam, M. J., Mouradov, D., Watt, S. J., Beck, J. L., Rodgers, K. J., Dean, R. T., Huber, T., Otting, G., and Dixon, N. E. (2005) Translational incorporation of L-3,4-dihydroxyphenylalanine into proteins. FEBS J. 272, 3162−3171. (28) Kigawa, T., Yabuki, T., Yoshida, Y., Tsutsui, M., Ito, Y., Shibata, T., and Yokoyama, S. (1999) Cell-free production and stable-isotope labeling of milligram quantities of proteins. FEBS Lett. 442, 15−19. (29) Gibbs, R. A. (1990) DNA amplification by the polymerase chain-reaction. Anal. Chem. 62, 1202−1214. (30) Stigers, D. J., Watts, Z. I., Hennessy, J. E., Kim, H. K., Martini, R., Taylor, M. C., Ozawa, K., Keillor, J. W., Dixon, N. E., and Easton, C. J. (2011) Incorporation of chlorinated analogues of aliphatic amino acids during cell-free protein synthesis. Chem. Commun. 47, 1839− 1841.

AUTHOR INFORMATION

Corresponding Authors

*E-mail: [email protected]. *E-mail: [email protected]. ORCID

Apostolos Alissandratos: 0000-0002-0177-3617 Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS The authors acknowledge funding from the Australian Research Council (grant number DP150101425).

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REFERENCES

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DOI: 10.1021/acschembio.6b00838 ACS Chem. Biol. XXXX, XXX, XXX−XXX