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Optimized incorporation of an unnatural fluorescent amino acid affords measurement of conformational dynamics governing high-fidelity DNA replication

2020; Elsevier BV; Volume: 295; Issue: 50 Linguagem: Inglês

10.1074/jbc.ra120.015557

1083-351X

Tyler L. Dangerfield, Kenneth A. Johnson,

RNA and protein synthesis mechanisms

DNA polymerase from bacteriophage T7 undergoes large, substrate-induced conformational changes that are thought to account for high replication fidelity, but prior studies were adversely affected by mutations required to construct a Cys-lite variant needed for site-specific fluorescence labeling. Here we have optimized the direct incorporation of a fluorescent un-natural amino acid, (7-hydroxy-4-coumarin-yl)-ethylglycine, using orthogonal amber suppression machinery in Escherichia coli. MS methods verify that the unnatural amino acid is only incorporated at one position with minimal background. We show that the single fluorophore provides a signal to detect nucleotide-induced conformational changes through equilibrium and stopped-flow kinetic measurements of correct nucleotide binding and incorporation. Pre-steady-state chemical quench methods show that the kinetics and fidelity of DNA replication catalyzed by the labeled enzyme are largely unaffected by the unnatural amino acid. These advances enable rigorous analysis to establish the kinetic and mechanistic basis for high-fidelity DNA replication. DNA polymerase from bacteriophage T7 undergoes large, substrate-induced conformational changes that are thought to account for high replication fidelity, but prior studies were adversely affected by mutations required to construct a Cys-lite variant needed for site-specific fluorescence labeling. Here we have optimized the direct incorporation of a fluorescent un-natural amino acid, (7-hydroxy-4-coumarin-yl)-ethylglycine, using orthogonal amber suppression machinery in Escherichia coli. MS methods verify that the unnatural amino acid is only incorporated at one position with minimal background. We show that the single fluorophore provides a signal to detect nucleotide-induced conformational changes through equilibrium and stopped-flow kinetic measurements of correct nucleotide binding and incorporation. Pre-steady-state chemical quench methods show that the kinetics and fidelity of DNA replication catalyzed by the labeled enzyme are largely unaffected by the unnatural amino acid. These advances enable rigorous analysis to establish the kinetic and mechanistic basis for high-fidelity DNA replication. The role of substrate-induced conformational changes in enzyme specificity has been a controversial topic for decades (1Roettger M.P. Bakhtina M. Tsai M.-D. Mismatched and matched dNTP incorporation by DNA polymerase β proceed via analogous kinetic pathways.Biochemistry. 2008; 47 (18717589): 9718-972710.1021/bi800689dCrossref PubMed Scopus (30) Google Scholar). In particular, the extraordinarily high fidelity of DNA replication by DNA polymerases has been the subject of studies in enzymology for many years (2Patel S.S. Wong I. Johnson K.A. Pre-steady-state kinetic analysis of processive DNA replication including complete characterization of an exonuclease-deficient mutant.Biochemistry. 1991; 30 (1846298): 511-52510.1021/bi00216a029Crossref PubMed Scopus (472) Google Scholar, 3Wong I. Patel S.S. Johnson K.A. An induced-fit kinetic mechanism for DNA replication fidelity: direct measurement by single-turnover kinetics.Biochemistry. 1991; 30 (1846299): 526-53710.1021/bi00216a030Crossref PubMed Scopus (344) Google Scholar). Paradoxically, high-fidelity polymerases make fewer mistakes and copy DNA at a much faster rate than low-fidelity DNA polymerases (1Roettger M.P. Bakhtina M. Tsai M.-D. Mismatched and matched dNTP incorporation by DNA polymerase β proceed via analogous kinetic pathways.Biochemistry. 2008; 47 (18717589): 9718-972710.1021/bi800689dCrossref PubMed Scopus (30) Google Scholar, 4Tsai Y.-C. Johnson K.A. A new paradigm for DNA polymerase specificity.Biochemistry. 2006; 45 (16893169): 9675-968710.1021/bi060993zCrossref PubMed Scopus (205) Google Scholar). Crystal structures of the T7 DNA polymerase (5Doublié S. Tabor S. Long A.M. Richardson C.C. Ellenberger T. Crystal structure of a bacteriophage T7 DNA replication complex at 2.2 A resolution.Nature. 1998; 391 (9440688): 251-25810.1038/34593Crossref PubMed Scopus (1103) Google Scholar), a model system for high-fidelity DNA replication, showed large structural changes in the enzyme upon nucleotide binding, as have other polymerases, raising the question of the contribution of this step to the enzyme's high fidelity. The original T7 DNA polymerase papers were published almost 30 years ago, measuring the correct incorporation (2Patel S.S. Wong I. Johnson K.A. Pre-steady-state kinetic analysis of processive DNA replication including complete characterization of an exonuclease-deficient mutant.Biochemistry. 1991; 30 (1846298): 511-52510.1021/bi00216a029Crossref PubMed Scopus (472) Google Scholar), misincorporation (3Wong I. Patel S.S. Johnson K.A. An induced-fit kinetic mechanism for DNA replication fidelity: direct measurement by single-turnover kinetics.Biochemistry. 1991; 30 (1846299): 526-53710.1021/bi00216a030Crossref PubMed Scopus (344) Google Scholar), and exonuclease proofreading reactions (6Donlin M.J. Patel S.S. Johnson K.A. Kinetic partitioning between the exonuclease and polymerase sites in DNA error correction.Biochemistry. 1991; 30 (1988042): 538-54610.1021/bi00216a031Crossref PubMed Scopus (182) Google Scholar). These papers provided valuable insights into the kinetic basis for the high DNA replication fidelity of T7 DNA polymerase but were flawed in the analysis of data regarding a prechemistry nucleotide-induced conformational change, based on the interpretation of an observed small thio-elemental effect. The notion that a small observed thio-elemental effect can be interpreted as evidence for the existence of a rate-limiting conformational change preceding chemistry has since been shown to be invalid because the magnitude of the elemental effect is largely dependent on the nature of the transition state and is smaller than previously expected (7Showalter A.K. Tsai M.-D. A reexamination of the nucleotide incorporation fidelity of DNA polymerases.Biochemistry. 2002; 41 (12186540): 10571-1057610.1021/bi026021iCrossref PubMed Scopus (126) Google Scholar, 8Wang H. Huang N. Dangerfield T. Johnson K.A. Gao J. Elber R. Exploring the reaction mechanism of HIV reverse transcriptase with a nucleotide substrate.J. Phys. Chem. B. 2020; 124 (32364738): 4270-428310.1021/acs.jpcb.0c02632Crossref PubMed Scopus (6) Google Scholar). The first direct measurement of a prechemistry conformational change came from studies on the low-fidelity repair enzyme, polymerase β, using a 2-aminopurine fluorescent reporter in the DNA (9Zhong X. Patel S.S. Werneburg B.G. Tsai M.D. DNA polymerase β: Multiple conformational changes in the mechanism of catalysis.Biochemistry. 1997; 36 (9305982): 11891-1190010.1021/bi963181jCrossref PubMed Scopus (99) Google Scholar, 10Zhong X. Patel S. Tsai M.D. DNA polymerase β: 5. Dissecting the functional roles of the two metal ions with Cr(III)dTTP.J. Am. Chem. Soc. 1998; 120: 235-23610.1021/ja973507rCrossref Scopus (30) Google Scholar, 11Dunlap C.A. Tsai M.-D. Use of 2-aminopurine and tryptophan fluorescence as probes in kinetic analyses of DNA polymerase β.Biochemistry. 2002; 41 (12220188): 11226-1123510.1021/bi025837gCrossref PubMed Scopus (95) Google Scholar). These studies showed that the conformational change was faster than chemistry, challenging previous notions that the conformational change had to be rate-limiting to contribute to fidelity. Critics of these studies, however, proposed that the fluorescence signal could be due to changes in the DNA structure and translation, rather than the conformational change in the enzyme. To directly measure the rate of the conformational change for the T7 DNA polymerase, an 8-Cys-lite T7 DNA polymerase variant was labeled with 7-diethylamino-3-((((2-maleimidyl)ethyl)amino)carbonyl)coumarin (MDCC) at a surface-exposed engineered cysteine in the highly mobile fingers domain (4Tsai Y.-C. Johnson K.A. A new paradigm for DNA polymerase specificity.Biochemistry. 2006; 45 (16893169): 9675-968710.1021/bi060993zCrossref PubMed Scopus (205) Google Scholar). By labeling the protein instead of the DNA and measuring the relevant rate constants, these studies confirmed that the rate of the conformational change was indeed faster than chemistry for both correct and mismatched nucleotides and discovered that the reverse of the conformational change was much slower than chemistry for the correct nucleotide but much faster than chemistry for the mismatched nucleotide, suggesting a new paradigm for understanding enzyme specificity (4Tsai Y.-C. Johnson K.A. A new paradigm for DNA polymerase specificity.Biochemistry. 2006; 45 (16893169): 9675-968710.1021/bi060993zCrossref PubMed Scopus (205) Google Scholar). However, the extensive mutagenesis required to create the 8-Cys-lite variant negatively affected the enzyme. This variant had low solubility and most importantly at least a 50-fold decrease in the apparent Kd for a mismatched nucleotide, reducing the enzyme's fidelity. Subsequent similar studies using HIV reverse transcriptase, where only a single cysteine had to be removed, confirmed the central role of the nucleotide-induced conformational change in DNA replication fidelity (12Kellinger M.W. Johnson K.A. Nucleotide-dependent conformational change governs specificity and analog discrimination by HIV reverse transcriptase.Proc. Natl. Acad. Sci. U. S. A. 2010; 107 (20385846): 7734-773910.1073/pnas.0913946107Crossref PubMed Scopus (68) Google Scholar). However, HIV reverse transcriptase has only moderate fidelity, so it is important to more accurately assess the role of conformational changes in specificity with a high-fidelity enzyme, which is all the more important given the controversies (13Ram Prasad B. Kamerlin S.C.L. Florián J. Warshel A. Prechemistry barriers and checkpoints do not contribute to fidelity and catalysis as long as they are not rate limiting.Theor. Chem. Acc. 2012; 131128810.1007/s00214-012-1288-6Crossref Scopus (27) Google Scholar). Because the T7 DNA polymerase is an ideal model system for a fast, high-fidelity enzyme, it was essential to improve upon strategies for labeling the enzyme with a fluorophore to report changes in enzyme structure. Here, we present the results of our optimization of the expression, purification, and basic kinetic characterization of a variant containing the fluorescent unnatural amino acid (7-hydroxy-4-coumarin-yl)-ethylglycine (7-HCou) on the surface of the fingers domain. We show that the fluorescence is sensitive to conformational changes of the protein during nucleotide incorporation (Fig. 1). By comparison to WT enzyme, we show that labeling of the enzyme with the fluorescent amino acid has negligible effects on polymerization kinetics and fidelity. By optimizing expression, the proteins could be purified without the use of added tags which also might alter enzyme activity. The methods in this paper overcome the limitations of the cysteine-maleimide labeling strategy and provide the groundwork for full characterization of the role of conformational dynamics of this enzyme on DNA replication fidelity and proofreading. Our optimized methods can be extended to other enzyme systems. Small fluorescence probes with quantum yields that are sensitive to local environment have significant advantages over larger probes commonly used in biochemistry, such as GFP, Cy3, quantum dots, and dual probes for FRET distance measurements. With a probe sensitive to local environment, there is no need to do traditional FRET experiments that require dual labeling with commensurate higher chances to interfere with enzyme function, because comprehensive global data fitting defines the step being measured by the fluorescence signal (14Johnson K.A. Kinetic analysis for the new enzymology: Using computer simulation to learn kinetics and solve mechanisms. KinTek Corporation, Austin, U. S. A2019Google Scholar, 15Kellinger M.W. Johnson K.A. Role of induced fit in limiting discrimination against AZT by HIV reverse transcriptase.Biochemistry. 2011; 50 (21548586): 5008-501510.1021/bi200204mCrossref PubMed Scopus (32) Google Scholar). The optical properties of 7-hydroxycoumarin in various solvents (16Zinsli P.E. Investigation of rate parameters in chemical reactions of excited hydroxycoumarins in different solvents.J. Photochem. 1974; 3: 55-6910.1016/0047-2670(74)80006-7Crossref Scopus (38) Google Scholar) and at different pH values (pKa ∼ 7.8 (17Fink D.W. Koehler W.R. pH effects on fluorescence of umbelliferone.Anal. Chem. 1970; 42: 990-99310.1021/ac60291a034Crossref Scopus (128) Google Scholar)) have been well-characterized and show that this fluorophore can be used to probe local environment. To circumvent the problems associated with the cysteine labeling strategy used for the 8-Cys-lite variant (18Tsai Y.-C. Jin Z. Johnson K.A. Site-specific labeling of T7 DNA polymerase with a conformationally sensitive fluorophore and its use in detecting single-nucleotide polymorphisms.Anal. Biochem. 2009; 384 (18834851): 136-14410.1016/j.ab.2008.09.006Crossref PubMed Scopus (14) Google Scholar), we tested the amber suppression to insert a site-specific unnatural amino acid, requiring only one residue substitution in the protein to directly incorporate a fluorescent unnatural amino acid into the fingers domain of the T7 DNA polymerase (Fig. 1). This unnatural amino acid system repurposes the amber stop codon (TAG) as the signal for insertion of an unnatural amino acid, using an evolved orthogonal Methanocaldococcus jannaschii tyrosyl-tRNA synthetase variant (aminoacyl tRNA synthetase; AARS) and M. jannaschii tyrosyl-tRNA with CUA in the anticodon loop (tRNAMj) expressed in E. coli cells. The amber stop codon is the least used of the three stop codons in E. coli (∼9%) and rarely terminates essential genes (19Xie J. Schultz P.G. An expanding genetic code.Methods. 2005; 36 (16076448): 227-23810.1016/j.ymeth.2005.04.010Crossref PubMed Scopus (110) Google Scholar). The M. jannaschii AARS is amenable to directed evolution to incorporate a wide variety of unnatural amino acids (4Tsai Y.-C. Johnson K.A. A new paradigm for DNA polymerase specificity.Biochemistry. 2006; 45 (16893169): 9675-968710.1021/bi060993zCrossref PubMed Scopus (205) Google Scholar). To implement this system with the T7 DNA polymerase, the codon for residue 514 in T7 gene 5 in pcIts-(T7 G5x−, trxA), described below, was mutated to an amber codon by site-directed mutagenesis to provide the codon for unnatural amino acid incorporation; the resulting plasmid was designated pcIts-(T7 G5x− E514TAG, trxA). This position was chosen because it was the amino acid position that gave the best signal using the cysteine-MDCC labeling strategy with the 8-Cys-lite T7 DNA polymerase variant (4Tsai Y.-C. Johnson K.A. A new paradigm for DNA polymerase specificity.Biochemistry. 2006; 45 (16893169): 9675-968710.1021/bi060993zCrossref PubMed Scopus (205) Google Scholar). We first worked to optimize the expression and purification of WT T7 DNA polymerase. Previous studies showed that coexpression of E. coli thioredoxin and T7 gene product 5 (GP5), the two polypeptides that together constitute the T7 DNA polymerase holoenzyme, greatly enhanced the solubility of a histidine-tagged T7 DNA polymerase variant (20Chiu J. Tillett D. March P.E. Coexpression of the subunits of T7 DNA polymerase from an artificial operon allows one-step purification of active gp5/Trx complex.Protein Expr. Purif. 2006; 47 (16300964): 264-27210.1016/j.pep.2005.10.016Crossref PubMed Scopus (6) Google Scholar). Al-though thioredoxin was constitutively expressed in E. coli, significant levels of overexpression of T7 GP5 required overexpression of the thioredoxin cofactor as well. We therefore constructed the plasmid pcIts-(T7 G5x−, trxA) (Fig. 2A), containing a bicistronic operon of T7 gene 5 (without a histidine tag) and trxA cloned into the pcIts backbone (21Brandis J.W. Johnson K.A. High-cell density shake-flask expression and rapid purification of the large fragment of Thermus aquaticus DNA polymerase I using a new chemically and temperature inducible expression plasmid in Escherichia coli.Protein Expr. Purif. 2009; 63 (18952180): 120-12710.1016/j.pep.2008.09.018Crossref PubMed Scopus (8) Google Scholar). The bicistronic operon contains a short linker between the genes for thioredoxin and T7 GP5, consisting of a PmeI restriction site followed by a ribosome binding site (Fig. 2B). The pcIts backbone includes a temperature-sensitive λ cIts repressor encoded on the plasmid that binds tightly to the operator sequence at 30 °C, blocking expression, but becomes inactive at temperatures greater than ∼35 °C. Having the repressor encoded by the plasmid allows expression in most E. coli strains, and no chemical inducer is required for high-level expression. We tested the utility of this system for expressing the T7 DNA polymerase in E. coli harboring the plasmid pcIts-(T7 G5x−, trxA). The cells were grown at 30 °C before heat induction at 42 °C for 20 min, followed by expression at 38 °C for 3 h, and were isolated by centrifugation. Cell pellets were later lysed and processed to obtain samples corresponding to the "Total Cell Protein," "Soluble Protein," and "Insoluble Protein" fractions at each time point. The gel in Fig. 2C shows soluble and highly overexpressed T7 DNA polymerase, easily seen above the other E. coli proteins. The procedure for purification of T7 DNA polymerase was based on further optimization of previous protocols (2Patel S.S. Wong I. Johnson K.A. Pre-steady-state kinetic analysis of processive DNA replication including complete characterization of an exonuclease-deficient mutant.Biochemistry. 1991; 30 (1846298): 511-52510.1021/bi00216a029Crossref PubMed Scopus (472) Google Scholar) to improve purity, increase the capacity of the method to accommodate the large increase in protein expression, and reduce the number of time-consuming dialysis steps. The optimized expression/purification protocol routinely yielded 50–100 mg of WT T7 DNA polymerase/liter of media. The purity and stoichiometry of the final product are shown in Fig. 2D, with T7 GP5 and thioredoxin copurifying in approximately a 1:1 ratio. To change the specificity of the M. jannaschii tyrosyl-tRNA synthetase (MjYRS) from tyrosine to 7-HCou, eight mutations were made in the AARS gene of the unnatural amino acid machinery plasmid pSUP-(MjYRS-6TRN), as described by Schultz and co-workers (22Summerer D. Chen S. Wu N. Deiters A. Chin J.W. Schultz P.G. A genetically encoded fluorescent amino acid.Proc. Natl. Acad. Sci. U. S. A. 2006; 103 (16785423): 9785-978910.1073/pnas.0603965103Crossref PubMed Scopus (211) Google Scholar).The resulting plasmid containing the mutant AARS was designated pSUP-(CouRS-6TRN) (Fig. 3A). Amber suppression efficiency at position 514 with this system was tested with E. coli harboring both pSUP-(CouRS-6TRN) and pcIts-(T7 G5x− E514TAG, trxA) (Fig. 4). As a control, a parallel culture was grown in the absence of 7-HCou to test for background incorporation of naturally occurring amino acids at position 514 in T7 GP5. No band for GP5 was observed in the lanes where 7-HCou was not added to the media (Fig. 4, lane 4), whereas a faint band appeared on the gel for the culture grown in the presence of 7-HCou (Fig. 4, lane 2), indicating low amber suppression efficiency. The literature contains many examples of optimization of various inefficient unnatural amino acid systems (23Young T.S. Ahmad I. Yin J.A. Schultz P.G. An enhanced system for unnatural amino acid mutagenesis in E. coli.J. Mol. Biol. 2010; 395 (19852970): 361-37410.1016/j.jmb.2009.10.030Crossref PubMed Scopus (470) Google Scholar, 24Liu J. Castañeda C.A. Wilkins B.J. Fushman D. Cropp T.A. Condensed E. coli cultures for highly efficient production of proteins containing unnatural amino acids.Bioorg. Med. Chem. Lett. 2010; 20 (20805030): 5613-561610.1016/j.bmcl.2010.08.049Crossref PubMed Scopus (10) Google Scholar), so we began testing a number of them to improve the yield of T7 DNA polymerase E514Cou.Figure 4Effect of optimized tRNA and condensed culture strategy on amber suppression efficiency. Lanes 1–4 contain samples from E. coli harboring pcIts-(T7 G5x− E514TAG, trxA) and pSUP-(CouRS-6TRN), analyzed by SDS-PAGE. Lanes 5–10 contain samples from E. coli harboring pcIts-(T7 G5x− E514TAG, trxA) and pSUP-(CouRS-tRNAopt), analyzed by SDS-PAGE. Samples with a + in the row labeled 10 × Cond. were condensed 10× by gentle centrifugation of the cells and resuspension into media with 1 mm 7-HCou before inducing protein expression. The row labeled 7-HCou indicates whether 7-HCou was added to the expression media (+), or not added to the expression media (−) to test for background incorporation of naturally occurring amino acids. The row labeled Ind. indicates whether the cells were collected before inducing protein expression (Pre) or 3 h after inducing protein expression (Post). The arrow to the right of the gel indicates the position of the band for T7 GP5 at ∼80 kDa.View Large Image Figure ViewerDownload Hi-res image Download (PPT) The first plasmid optimization was designed to test the effect of an amber suppressor tRNA variant of tRNAMj evolved to improve unnatural amino acid incorporation efficiency (25Guo J. Melançon C.E. Lee H.S. Groff D. Schultz P.G. Evolution of amber suppressor tRNAs for efficient bacterial production of proteins containing nonnatural amino acids.Angew. Chem. Int. Ed. Engl. 2009; 48 (19856359): 9148-915110.1002/anie.200904035Crossref PubMed Scopus (123) Google Scholar). Cells expressing six copies of tRNAMj from pSUP-(CouRS-6TRN) grew very slowly and did not reach the same stationary phase density as cells expressing the WT enzyme. As previously reported by others (23Young T.S. Ahmad I. Yin J.A. Schultz P.G. An enhanced system for unnatural amino acid mutagenesis in E. coli.J. Mol. Biol. 2010; 395 (19852970): 361-37410.1016/j.jmb.2009.10.030Crossref PubMed Scopus (470) Google Scholar), one of the tricistronic operons was occasionally lost during cloning steps, presumably from homologous recombination because of the adjacent, identical tRNA operons, resulting in a mixture of cells with different numbers of copies of tRNAMj. Substituting one copy of an optimized tRNAMj, designated tRNAopt (25Guo J. Melançon C.E. Lee H.S. Groff D. Schultz P.G. Evolution of amber suppressor tRNAs for efficient bacterial production of proteins containing nonnatural amino acids.Angew. Chem. Int. Ed. Engl. 2009; 48 (19856359): 9148-915110.1002/anie.200904035Crossref PubMed Scopus (123) Google Scholar), for six copies of tRNAMj has been successful with other unnatural amino acid systems and is reported to reduce the toxicity (25Guo J. Melançon C.E. Lee H.S. Groff D. Schultz P.G. Evolution of amber suppressor tRNAs for efficient bacterial production of proteins containing nonnatural amino acids.Angew. Chem. Int. Ed. Engl. 2009; 48 (19856359): 9148-915110.1002/anie.200904035Crossref PubMed Scopus (123) Google Scholar). The optimized tRNA was inserted in place of the six copies of tRNAMj with ligation-independent cloning (26Aslanidis C. de Jong P.J. Ligation-independent cloning of PCR products (LIC-PCR).Nucleic Acids Res. 1990; 18 (2235490): 6069-607410.1093/nar/18.20.6069Crossref PubMed Scopus (934) Google Scholar), and the resulting plasmid containing a single copy of tRNAopt is designated pSUP-(CouRS-tRNAopt) (Fig. 3B). Expression tests of E. coli harboring both pcIts-(T7 G5x-E514TAG, trxA) and pSUP-(CouRS-tRNAopt) show a large improvement in expression over the pSUP-(CouRS-6TRN) plasmid (Fig. 4, lane 2 versus lane 6). Additionally, no significant band was detected for the T7 DNA polymerase in the cultures without 7-HCou added to the media (Fig. 4, lane 4 versus lane 8), indicating that CouRS appears to be inefficient however specific for 7-HCou over endogenous amino acids. Cells harboring pSUP-(CouRS-tRNAopt) grew much faster and to higher densities than cells harboring pSUP-(CouRS-6TRN) and were not prone to loss of one of the operons. Other studies have found that an inducible copy of the AARS gene, combined with the constitutive copy from the mutant promoter for the E. coli glutamine-tRNA ligase gene (glnS') (27Ryu Y. Schultz P.G. Efficient incorporation of unnatural amino acids into proteins in Escherichia coli.Nat. Methods. 2006; 3 (16554830): 263-26510.1038/nmeth864Crossref PubMed Scopus (233) Google Scholar), can enhance amber suppression efficiency (23Young T.S. Ahmad I. Yin J.A. Schultz P.G. An enhanced system for unnatural amino acid mutagenesis in E. coli.J. Mol. Biol. 2010; 395 (19852970): 361-37410.1016/j.jmb.2009.10.030Crossref PubMed Scopus (470) Google Scholar, 28Cellitti S.E. Jones D.H. Lagpacan L. Hao X. Zhang Q. Hu H. Brittain S.M. Brinker A. Caldwell J. Bursulaya B. Spraggon G. Brock A. Ryu Y. Uno T. Schultz P.G. et al.In vivo incorporation of unnatural amino acids to probe structure, dynamics, and ligand binding in a large protein by nuclear magnetic resonance spectroscopy.J. Am. Chem. Soc. 2008; 130 (18576636): 9268-928110.1021/ja801602qCrossref PubMed Scopus (139) Google Scholar). To test the effect of an inducible copy of CouRS in our system, we added a copy of the gene under control of the λ promoter. Because the T7 DNA polymerase genes are also expressed under this promoter, supplemental expression of CouRS occurs simultaneously upon heat induction. This plasmid, pSUP-(2×CouRS-tRNAopt) (Fig. 5A), contains a single copy of tRNAopt, p15A origin of replication, CmR gene, and two copies of CouRS. This construct was tested for T7 DNA polymerase E514Cou expression, as above. An enhancement in amber suppression efficiency was observed, so that a significant band of the expected molecular mass appeared in the post-induction samples with 7-HCou added to the expression media (Fig. 5B, lane 2 versus lane 7). One final CouRS optimization was attempted by making the D286R mutation in CouRS (not shown). Some unnatural amino acid systems have shown this mutation to further enhance amber suppression (29Kobayashi T. Nureki O. Ishitani R. Yaremchuk A. Tukalo M. Cusack S. Sakamoto K. Yokoyama S. Structural basis for orthogonal tRNA specificities of tyrosyl-tRNA synthetases for genetic code expansion.Nat. Struct. Biol. 2003; 10 (12754495): 425-43210.1038/nsb934Crossref PubMed Scopus (173) Google Scholar); however, we found that this mutation did not significantly enhance the efficiency of our system. The plasmid pSUP-(2×CouRS-tRNAopt) was therefore used for all further expressions. Some optimization strategies were tested that did not require any DNA manipulation. Expression tests on the WT enzyme comparing rich media (Terrific Broth) with a supplemented M9 media (see "Experimental procedures") showed slightly higher specific expression of T7 DNA polymerase with the minimal media (not shown). This has the added advantage of greatly reducing the concentration of naturally occurring amino acids in the media, therefore reducing the likelihood of mis-acylation of the amber suppressor tRNA. The supplemented M9 media was used for all expression tests and large-scale expression of the T7 DNA polymerase E514Cou variant. A high concentration of unnatural amino acid (≥1 mm) is generally added to the media to have a high enough concentration inside the cells for incorporation into the target protein. Generally, only a small fraction is actually translated into proteins and the rest is lost in the media after the cells have been harvested. A strategy commonly used in the NMR and structural biology fields to minimize amounts of probe needed is the use of a condensed culture (24Liu J. Castañeda C.A. Wilkins B.J. Fushman D. Cropp T.A. Condensed E. coli cultures for highly efficient production of proteins containing unnatural amino acids.Bioorg. Med. Chem. Lett. 2010; 20 (20805030): 5613-561610.1016/j.bmcl.2010.08.049Crossref PubMed Scopus (10) Google Scholar). The condensed culture strategy involves growing large quantities of cells in standard media, gently pelleting them and resuspending in a much smaller volume of media containing the unnatural amino acid before inducing expression. In principle, the concentration is still high enough for efficient incorporation, although the yield of pellet with protein of interest can be increased greatly. When tested in our optimized plasmid system, a band of similar intensity, as compared with the uncondensed culture, appeared in the postinduction sample (Fig. 4, lane 6 versus lane 10), even at high cell densities. We therefore used this strategy in combination with the plasmid-based optimizations for large-scale expression of T7 DNA polymerase E514Cou. Large-scale protein expression was performed by inoculating six 1-liter cultures of supplemented M9 media with E. coli harboring pSUP-(2×CouRS-tRNAopt) and pcIts-(T7 G5x− E514TAG, trxA). The cultures were grown to an A600 of 0.5, and then the cells were gently pelleted and resuspended in 600 ml of supplemented M9 media containing 1 mm 7-HCou. Protein expression was induced by elevating the culture temperature from 30 to 42 °C, incubating for 30 min, and then dropping the temperature to 38 °C and incubating for 3 h. The T7 DNA polymerase E514Cou variant and the WT enzyme were purified with an optimized purification protocol to increase the capacity and reduce the time required to purify the protein, as described for WT enzyme. The presence of 7-HCou in the protein simplified the chromatography steps of the purification because it provided a convenient optical signature that could be followed by monitoring absorbance at 335 nm on the HPLC. Purification from a pellet from the 600-ml condensed culture yielded 5–10 mg