an enhanced system for unnatural amino acid mutagenesis in e. … · 362 a system for unnatural...

doi:10.1016/j.jmb.2009.10.030 J. Mol. Biol. (2010) 395, 361–374

Available online at www.sciencedirect.com

An Enhanced System for Unnatural Amino AcidMutagenesis in E. coli

Travis S. Young, Insha Ahmad, Jun A. Yin and Peter G. Schultz⁎

Department of Chemistry andthe Skaggs Institute forChemical Biology, The ScrippsResearch Institute, 10550 NorthTorrey Pines Road, La Jolla,CA 92037, USA

Received 8 July 2009;received in revised form6 October 2009;accepted 14 October 2009Available online21 October 2009

*Corresponding author. E-mail [email protected] used: aaRS, aminoa

(s); aaRS/tRNACUA, aminoacyl-tRNsuppressor tRNA; IR, infrared; GFPprotein; Myo, sperm whale myoglobnylalanine; OD600, optical density atp-azidophenylalanine; pBpF, p-benzpPrF, p-propargyloxyphenylalanine;phenyllactic acid; pIF, p-iodophenylp-cyanophenylalanine; pCmF, p-carblalanine; NapA, 3-(2-naphthyl)alaninp-boronophenylalanine; oNiF, o-nitroHQA, (8-hydroxy-quinolin-3-yl)alanbipyridin-5-yl)alanine; CouA, (7-hydethylglycine; EF-Tu, elongation factocentrifugal force.

0022-2836/$ - see front matter © 2009 E

We report a new vector, pEVOL, for the incorporation of unnatural aminoacids into proteins in Escherichia coli using evolved Methanocaldococcusjannaschii aminoacyl-tRNA synthetase(s) (aaRS)/suppressor tRNA pairs.This new system affords higher yields of mutant proteins through the useof both constitutive and inducible promoters to drive the transcription oftwo copies of the M. jannaschii aaRS gene. Yields were further increased bycoupling the dual-aaRS promoter system with a newly optimized sup-pressor tRNACUA

opt in a single-vector construct. The optimized suppressortRNACUA

opt afforded increased plasmid stability compared with previouslyreported vectors for unnatural amino acid mutagenesis. To demonstrate theutility of this new system, we introduced 14 mutant aaRS into pEVOL andcompared their ability to insert unnatural amino acids in response to threeindependent amber nonsense codons in sperm whale myoglobin or greenfluorescent protein. When cultured in rich media in shake flasks, pEVOLwas capable of producing more than 100 mg/L mutant GroEL protein.The versatility, increased yields, and increased stability of the pEVOLvector will further facilitate the expression of proteins with unnatural aminoacids.

© 2009 Elsevier Ltd. All rights reserved.

Keywords: unnatural amino acid; Escherichia coli; protein mutagenesis;optimized suppressor tRNA; orthogonal tRNA/aminoacyl-tRNA synthetase
Edited by J. Karn
Introduction

The ability to genetically encode unnatural aminoacids directly in living cells in response to nonsenseor frameshift codons allows one to manipulate the

ress:

cyl-tRNA synthetaseA synthetase/, green fluorescentin; pAcF, p-acetylphe-600 nm; pAzF,

oylphenylalanine;PLA, p-hydroxy-L-a-lanine; pCNF,oxylmethylpheny-e; pBoF,phenylalanine;ine; BipyA, (2,2'-roxycou-marin-4-yl)r Tu; rcf, relative

lsevier Ltd. All rights reserve

physiochemical, biological, and pharmacologicalproperties of proteins with exquisite control overstructure. This approach has a number of potentialadvantages over other “chemical mutagenesis”methods such as the use of chemically aminoacy-lated tRNAs, solid-phase peptide synthesis, nativechemical ligation, or intein-based semisyntheticmethods.1–5 In particular, this method can be usedto modify proteins directly in living cells, isapplicable to virtually any protein expressed inprokaryotic or eukaryotic organisms, affords excel-lent yields of mutant protein in general, and allows alarge number of chemically distinct amino acids tobe introduced site specifically by simple mutagen-esis. Unnatural amino acids have been selectivelyincorporated into proteins recombinantly expressedin Escherichia coli,6 Saccharomyces cerevisiae,7 Pichiapastoris,8 and mammalian cells9–11 by reassigningamber nonsense (TAG) or frameshift codons toencode the desired unnatural amino acid. Orthog-onal aminoacyl-tRNA synthetase/suppressor tRNA(aaRS/tRNACUA) pairs are used to selectivelyincorporate the unnatural amino acid. These or-thogonal aaRS aminoacylate their cognate tRNAs,

d.

mailto:[email protected]://dx.doi.org/10.1016/j.jmb.2009.10.030

362 A System for Unnatural Amino Acid Mutagenesis

but not any of the host organism's endogenoustRNAs; similarly, the orthogonal tRNA is notaminoacylated by any endogenous aaRS. This lackof crossreactivity ensures that the unnatural aminoacid is incorporated with high fidelity only inresponse to the nonsense or frameshift codon12 andthat no endogenous amino acid is encoded inresponse to these codons. To alter the specificity ofan aaRS in order to aminoacylate its cognate tRNAwith only the desired unnatural amino acid (andno endogenous amino acid), we subjected mutantlibraries centered on the amino acid binding pocket torounds of positive and negative selections based onsuppression of the amber codon at permissive sites inessential or lethal gene products, respectively.13

The genetic incorporation of unnatural aminoacids into proteins in E. coli in this manner has beencarried out using orthogonal pairs derived fromMethanosarcina mazei,14 Pyrococcus horikoshii,10 andother archaeal systems, but has been most practicedusing Methanocaldococcus jannaschii tyrosyl-RS/tRNACUA

Tyr derived pairs.15,16 This system has beenexploited to incorporate more than 30 unnaturalamino acids, including metal ion binding,17

fluorescent,18 photocrosslinking, and photocaged19

amino acids; amino acids containing NMR,20,21infrared (IR),22 and crystallographic23 probes; andamino acids with orthogonal chemical reactivity,24

among others. Although high yields (up to 1 g/L inhigh-density fermentation) of proteins can begenerated by these methods, there remain contexteffects25,26 and varied efficiencies for the incorpora-tion of unnatural amino acids by specific aaRS thatcan result in variable yields of some mutantproteins. Some systems also rely on minimalmedia to exclude the incorporation of endogenousamino acids by the unnatural aaRS and lack stabilityunder some growth conditions.It has previously been reported by Cellitti et al.

that inducible control of aaRS allows an increasedexpression of mutant proteins containing unnaturalamino acids.21 Moreover, we have recently identi-fied specific mutations in the M. jannaschii suppres-sor tRNA that afford an increased expression ofmutant proteins in E. coli. In an effort to bothstandardize the expression system used for unnat-ural amino acid mutagenesis and achieve increasedyields of mutant proteins, the pEVOL system, whichuses an inducible and constitutive aaRS system witha single copy of the novel tRNA, was created. Thissystem was optimized by analyzing the effects oftRNA copy number, aaRS induction parameters,and expression temperature on yields. In a 0.5-Lshake flask expression of the chaperonin proteinGroEL, pEVOL afforded N100 mg/L mutant proteinin either DH10B or BL21 E. coli. Furthermore, 14unnatural aaRS previously evolved from the M.jannaschii tyrosyl-RS were inserted into the pEVOLsystem and compared in terms of their efficiency toincorporate unnatural amino acids in response to anamber nonsense codon at three distinct sites in greenfluorescent protein (GFP) or sperm whale myoglo-bin (Myo).

Results

General construction of pEVOL

The pEVOL series of plasmids was constructedbased on previous studies that showed an increasedexpression of the orthogonal aaRS/tRNACUA

Tyr pairwas effective in decreasing protein truncation and inincreasing overall yields of mutant proteins.21,27 Aplasmid with a mid-copy-number to low-copy-number p15A origin27,28 was chosen to encode theaaRS/tRNACUA

Tyr pair. This origin allows target genesof interest to be harbored in compatible colE1 orpBR322 expression plasmids. To allow a rapidassessment of protein yields in order to optimizethe expression system, we inserted the gene for GFPwith a Tyr151TAGmutation under the control of theT7 promoter in a pET101 vector (pET-GFPY151X). Inthis case, fluorescence is observed only when theTAG codon acts as a sense codon for the aminoa-cylated suppressor tRNACUA and not as a stopcodon to afford GFP truncated at 150 amino acids.The yield of GFPY151X is, in general, significantlylower than those of other mutant proteins contain-ing unnatural amino acids, providing a good systemfor vector optimization. Fluorescence intensitieswere measured in the presence or in the absence ofunnatural amino acid to ensure that full-lengthfluorescent GFP was a consequence of unnaturalamino acid incorporation. Selective incorporation ofthe p-acetylphenylalanine (pAcF) amino acid bypAcF-RS was further confirmed by mass spectrom-etry (vide infra). T7 RNA polymerase, which drivesGFP transcription in pET101, was expressed from aDE3 λ prophage under an isopropyl β-D-1-thioga-lactopyranoside (IPTG)-inducible lacI promoter inBL21(DE3). The relative efficiency of unnaturalamino acid incorporation was measured by com-paring the fluorescence of E. coli cells transformedwith pEVOL-aaRS/pET-GFPY151X to that of wild-type GFP (pEVOL-aaRS/pET-GFPWT) and normal-ized to culture density, as described inMaterials andMethods. To assess variations in expression yieldsarising from the site of mutation or the nature of thetarget protein, we also suppressed three indepen-dent nonsense mutations in the Myo gene (MyoS4X,MyoF107X, and MyoY152X) under the control of the T7promoter in pET101. A 6×His tag was fused to the C-terminus of myoglobin and is only present when theTAG codon is suppressed by an unnatural aminoacid, allowing for affinity purification of the mutantmyoglobin.

Optimization of tRNA and aaRS copy number

To test whether constitutive or inducible expres-sion of the aaRS increases mutant protein yields, weinserted a modified glnS′ (constitutive)27,29 oraraBAD (arabinose-inducible) promoter30 5′ of theaaRS gene (Fig. 1a). The aaRS specific for pAcF(pAcF-RS) was chosen as a model due to its highfidelity and widespread use.31 The initial pEVOL

Fig. 1. DNA constructs and tRNA sequences in pEVOL. (a) The aaRS gene (orange) was placed under the control of thearaBAD promoter and the rrnB terminator (maroon) derived from pBAD (top line) or between the glnS′ promoter and theglnS T terminator (maroon) (bottom line). (b) tRNACUA

Tyr or tRNACUAopt (green) was placed under the control of the proK

promoter (prom; maroon) and the proK terminator (term; maroon) as a monocistronic, bicistronic, or tricistronic cluster(no bicistronic version was created for tRNACUA

Tyr ). Endogenous E. coli valU-valX or ileT-alaT linker sequences are indicatedby blue lines. (c) Cloverleaf depiction of tRNACUA

opt (left) and tRNACUATyr (right) showing the mutated anticodon (blue) and

the optimized T-stem sequence (red). (d) The final pEVOL construct combines both promoters (maroon), a monocistronictRNACUA

opt (tRNA; green), a p15A origin of replication (p15A; orange), the chloramphenicol acetyltransferase marker(CmR; maroon), and the araC repressor gene (araC; green).

363A System for Unnatural Amino Acid Mutagenesis

plasmid (pEVOL-a) encoding the pAcF-RS genedownstream of the glnS′ promoter produced lowyields of mutant protein when cotransformed withpET-GFPY151X into BL21(DE3) cells (Fig. 2a). Substi-tution of the glnS′ promoter and terminator with theinducible araBAD promoter and rrnB terminator(pEVOL-b) resulted in increased GFPY151X mutantprotein expression, but yields were still onlyapproximately 5–10% of those of wild-type GFP(pET-GFPWT) based on relative GFP fluorescence.Both constructs also yielded small amounts ofmutant myoglobin, as evidenced by faint bands ona Coomassie-stained SDS-PAGE gel (Fig. 2d).Recently, Cellitti et al. used a dual glnS′/araBADpromoter system in their pSUPAR3 construct, withgood success.21 Therefore, a similar construct, whichemploys both the araBAD promoter and the glnS′promoter (pEVOL-c), was created (Fig. 1d). Cotrans-formation of the dual-promoter construct with pET-GFPY151X yielded roughly 20% GFPY151X mutantprotein relative to GFPWT based on fluorescence.Use of both promoters may increase yields bycreating a basal level of aaRS for aminoacylation ofthe suppressor tRNA at the start of induction andincreased levels of the aaRS, as needed over thecourse of protein expression.Next, the effects of single or multiple copies of the

tRNACUATyr gene on mutant protein yields were deter-

mined with the pEVOL system. Previous work fromour laboratory showed that tRNACUA

Tyr genes ar-

ranged in polycistronic constructs separated byendogenous E. coli linker sequences (valU-valX orileT-alaT) can be effective at increasing the efficiencyof unnatural amino acid incorporation27 (Fig. 1b).Polycistronic suppressor tRNACUA cassettes havealso increased protein yields in yeast and mamma-lian cells.13 Furthermore, the proK promoter wasshown to be highly successful at driving thetranscription of monocistronic and polycistronictRNACUA

Tyr constructs.27 However, integration ofone, two, or three copies of the suppressor tRNACUA

Tyr

in pEVOL (pEVOL-c, pEVOL-d, and pEVOL-e,respectively) did not appear to significantly increasethe yield of the mutant GFPY151X (Fig. 2a). Similarly,little change was observed in the yield of all threemyoglobin mutants by altering tRNACUA

Tyr copynumber (Fig. 2d).

Effects of tRNA and aaRS mutations

Directed evolution experiments that have focusedon the T-stem of tRNACUA

Tyr recently identified amodified suppressor (tRNACUA

opt ) that increasedunnatural amino acid incorporation efficiencywith several aaRS (Fig. 1c).32 To test whether thisoptimized tRNA would increase protein expressionin the pEVOL system, we inserted tRNACUA

opt down-stream of the proK promoter as a monocistronicor tricistronic cluster in an identical fashion totRNACUA

Tyr . Both one and three copies of tRNACUAopt in

Fig. 2. pET-GFPY151X and pET-Myo expressions with pEVOL constructs. (a) Constructs containing one, two, orthree copies of tRNACUA

Tyr (Tyr) or tRNACUAopt (opt) and the pAcF-RS under the control of glnS′ (glnS'), araBAD (araB),

or both (dual) were analyzed for their ability to suppress an amber mutant at position 151 in GFP in the presence(blue) or in the absence (red) of 1 mM pAcF. In one constructs, pAcF-RS was replaced by the wild-type tyrosyl-RS(Tyr), as indicated. Residue 286 (Res 286) was left as wild-type Asp (D) or mutated to Arg (R), as indicated.Fluorescence was expressed as a percentage of pEVOL-aaRS cotransformed with a wild-type pET-GFPWT. Error bars,SD (n=3). (b) The pBK expression system (pBK-pAcF/pLeiGY151X) was expressed under identical conditions in thepresence (blue) and in the absence (no fluorescence) of 1 mM pAcF and compared with wild-type GFP from pBK-aaRS/pLeiGWT. Error bars, SD (n=3). (c) The pSUP expression system (pSUP-pAcF/pET-GFPY151X) was expressedunder identical conditions and compared with wild-type GFP from pSUP-aaRS/pET-GFPWT. Error bars, SD (n=4).(d) Three nonsense mutants were independently suppressed in Myo (MyoS4X, top; MyoF107X, middle; MyoY152X,bottom) using the pEVOL constructs listed above the gels. The Tyr-RS construct was only expressed with MyoS4X.Gels show affinity-purified 6×His-tagged myoglobin from 10 ml of BL21(DE3) cultures analyzed with a denaturingSDS-PAGE gel and stained with Coomassie brilliant blue. For comparison, the final lane in the top gel shows wild-type protein yields from pEVOL-aaRS/pET-MyoWT.


pEVOL (pEVOL-f and pEVOL-g, respectively) in-creased the yield of the GFPY151X mutant toapproximately 50% of the level of GFPWT basedon relative fluorescence intensities (Fig. 2a). Thetricistronic tRNACUA

opt also improved the yields of allthree myoglobin mutants (Fig. 2d). To test the noveltRNA with a different aaRS, we inserted the wild-type Tyr-RS into the pEVOL-f construct to createpEVOL-Tyr and assayed it with pET-GFPY151X andpET-MyoS4X. The yield of the GFPY151X mutant wasapproximately 75% that of GFPWT with Tyr-RSbased on relative fluorescence. Protein yields frompEVOL-Tyr/pET-MyoS4X were similarly high (Fig.2d), suggesting that this system may be generallyuseful (vide infra).The amino acid mutation D286R had been

previously shown by Kobayashi et al. to increasetRNACUA

Tyr anticodon (CUA) recognition by the M.jannaschii tyrosyl-RS.33 This mutation was alsodemonstrated in our laboratory to be advantageouswith p-benzoylphenylalanyl-RS.27 To evaluate theeffects of D286R on the pEVOL system, we intro-duced this mutation into the pAcF-RS gene in the

pEVOL construct containing one copy of tRNACUAopt

(pEVOL). Although the yield of the GFPY151Xmutant was not significantly different from thatof pEVOL harboring the monocistronic tRNACUA

opt

pEVOL-pAcF lacking D286R (pEVOL-f; Fig. 2a),protein yields were increased among all threemyoglobin mutants and were comparable to yieldsobtained with pEVOL harboring three copies oftRNACUA

opt (Fig. 2d).

Comparison of pEVOL with other expressionsystems

To compare the pEVOL system to previouslydeveloped unnatural amino acid incorporationsystems, we inserted the pAcF-RS gene into twopreviously reported vectors, pBK31 and pSUP,27 andused them to incorporate pAcF into GFPY151X. ThepBK vector harbors one copy of the pAcF-RS geneunder the native glnS promoter. For expres-sion experiments, one copy of the tRNACUA

Tyr gene(under the lpp promoter) is encoded along withGFPY151X in an orthogonal pLei expression plasmid

Fig. 3. Growth curves of (a) DH10B or (b) BL21(DE3) E.coli transformed with pEVOL constructs. Constructsharbored a single copy of tRNACUA

Tyr (Tyr), a single copyof tRNACUA

opt (opt), or a random DNA sequence with notRNA (control) between the proK promoter and the proKterminator. Cultures were grown at 37 °C for 8 h in 2×YTmedia supplemented with chloramphenicol and moni-tored by absorbance at 600 nm. Relative cell densities areindicated for 200-μl cultures and are an average of fourindependent cultures.


(pLeiGY151X).34 The pSUP vector encodes one copy

of the pAcF-RS gene (under a modified glnS′promoter27) and two tricistonic clusters of thetRNACUA

Tyr gene under the proK promoter. pBK-pAcFwas cotransformed with pLeiGY151X or pLeiGWT intoDH10B E. coli, and pSUP-pAcF was cotransformedwith pET-GFPY151X or pET-GFPWT into BL21(DE3)E. coli. The pBK-pAcF/pLeiGY151X and pSUP-pAcF/pET-GFOY151X systems afforded approximately 5%and 20% the yields of mutant GFPY151X relative toGFPWT, respectively, based on fluorescence intensi-ties (Fig. 2b and c). This is consistent with previousreports suggesting that pSUP increases the efficiencyof unnatural amino acid incorporation comparedwith pBK.27 The pEVOL vector further increasedyields to roughly 250% that of the previouslyoptimized pSUP.

Effects of pEVOL on E. coli growth rates

Overexpression of M. jannaschii tRNACUATyr (with

strong promoters such as proK) exhibits a slowedgrowth phenotype in E. coli, whereas tRNACUA

opt

appears to have a significantly lower effect ongrowth rates. To assess these effects on distinctE. coli strains that might be used for protein expres-sion, we transformed E. coli DH10B or BL21 withpEVOL containing a single copy of tRNACUA

Tyr

(pEVOL-c) or a single copy of tRNACUAopt (pEVOL).

Single colonies were picked, and growth in 2×YTmedia was monitored by optical density at 600 nm(OD600) (Fig. 3). Growth rates of pEVOL-c or pEVOLwere compared to a control construct created byinserting a region of DNA lacking a tRNA codingregion between the proK promoter and the proKterminator. The specific growth rates (μ) for controlsin DH10B and BL21 were 0.63 h−1 and 0.76 h−1,respectively (see Materials and Methods for details).Transformation with pEVOL-c decreased the spe-cific growth rate by 28.6% in DH10B (μ=0.45 h−1)and by 43.2% in BL21 (μ=0.43 h−1). Use of pEVOLslightly improved the growth rate in DH10B(μ=0.48 h−1) (Fig. 3a), but increased the growthrate in BL21 (μ=0.72 h−1), to near-wild-type growthrates (Fig. 3b). Increasing the copy number of eithertRNA increased toxicity in both strains (datanot shown) and resulted in unstable plasmidsthat were susceptible to a spontaneous loss ofportions of the tRNA region. For this reason, thefinal pEVOL construct (Fig. 1d) encoded only onecopy of tRNACUA

opt .

Optimization of mutant protein expressionconditions

The pEVOL/pET system in BL21(DE3) E. coliallows independent transcriptional regulation ofthe target gene of interest (IPTG) and the aaRS(araBAD). The effects of the degree, timing, andtemperature of the induction of these genes onmutant protein yields were therefore assessedusing pEVOL-pAcF/pET-GFPY151X cotransformedin BL21(DE3) E. coli. Cells were grown to

OD600=0.5–0.7, induced with arabinose (0.0002–2%) or IPTG (0.01–100 mM), and grown for 12–14 hat 30 °C. Induction with 0.02% arabinose and 1 mMIPTG yielded maximal mutant protein based onfluorescence (just over 50% compared with pET-GFPWT induced with 1 mM IPTG) (Fig. 4a). Theyield of GFPY151X dropped sharply when less than0.02% arabinose was used, consistent with theautocatalytic (or all-or-nothing) mechanism ofarabinose induction.35 Other concentrations ofinducers were less effective.To test whether it was necessary to induce

araBAD-aaRS prior to induction of the T7 polymer-ase, we independently varied arabinose and IPTGfrom early induction (OD600=0.2) to mid induction(OD600=0.7) or late induction (OD600=1.4). Theunnatural amino acid pAcF (1 mM) was added atthe time of arabinose induction. Maximal GFPY151Xyields (N50% of pET-GFPWT, which was induced atOD600=0.7) were observed when both araBAD-pAcF-RS and T7 polymerase were simultaneouslyinduced in late growth phase (OD600=1.4) at 30 °C(Fig. 4b, left). Induction of araBAD-aaRS prior toinduction of the T7 polymerase was marginally less

Fig. 4. Optimization of induction parameters. (a) BL21(DE3) cotransformed with pEVOL-pAcF/pET-GFPY151X wasgrown to OD600=0.6–0.8 and simultaneously induced with arabinose (Arab) and IPTG. Arabinose concentration wasvaried from 0.0002% to 2% or none, and IPTG concentration was varied from 0.01 mM to 100 mM or none.Fluorescence is shown relative to pEVOL-aaRS/pET-GFPWT induced with 1 mM IPTG. Error bars, SD (n=3). (b)Induction order was varied by inducing B21(DE3) (pEVOL-pAcF/pET-GFPY151X) with either 0.02% arabinose or 1 mMIPTG at an OD600 of 0.2, 0.7, or 1.4. Cells were induced in the presence (+pAcF) or in the absence (−pAcF) of 1 mMpAcF amino acid. Fluorescence is shown relative to pEVOL-aaRS/pET-GFPWT induced at an OD600 of 0.7. Error bars,SD (n=4). (c) The effects of temperature on amber suppression were assayed by expressing pEVOL-pAcF/pET-GFPY151X (pAcF) or pEVOL-aaRS/pET-GFPWT (WT) at 23 °C, 30 °C, or 37 °C. The scale represents relative fluo-rescence. Error bars, SD (n=3).


effective; however, T7 polymerase induction priorto induction of araBAD-aaRS significantly de-creased GFPY151X yields to below 20% of those ofpET-GFPWT. Similar experiments in the absence ofpAcF confirmed that these results are a conse-quence of unnatural amino acid incorporation (Fig.4b, right).To assess whether temperature has an effect on the

efficiency of unnatural amino acid incorporation, wetransformed BL21(DE3) E. coli with pEVOL-pAcF/pET-GFPY151X or pEVOL-aaRS/pET-GFPWT andgrew them for 12–14 h at 23 °C, 30 °C, or 37 °C.Induction was carried out as discussed above, andincorporation of pAcF was measured at eachtemperature. Although overall maximal yieldswere observed at 30 °C based on fluorescence, theyield of mutant GFPY151X was roughly 50% that ofGFPWT, regardless of temperature (Fig. 4c). Thisindicates that unnatural amino acid incorporationwith pEVOL has little dependence on temperatureover this range and may be adaptable to a widevariety of protein expression protocols.

Expression of a model protein (GroEL) underoptimized conditions

Todemonstrate the utility of this optimized systemin larger-scale (0.5 L) expression experiments, wecompared pEVOL-pAcF with pSUP-pAcF and pBK-pAcF for its ability to insert the pAcF unnaturalamino acid in response to a TAGcodon atposition 129in the E. coli chaperonin protein GroEL (GroELE129X).To express GroELE129X with pEVOL or pSUP, weused the IPTG-inducible pTrc plasmid to harbor theGroELE129X gene.

36 To allow compatibility with thepBK plasmid, we used the pBADJY vector (encodingthe tRNACUA

Tyr gene, as described by Zhang et al.6 andWang et al.31) to encode GroELE129X. Plasmids werecotransformed into BL21 or DH10B E. coli, andprotein expression was carried out at 37 °C for 10 hor 20 h using optimized induction parameters.Examination of crude cell lysate by SDS-PAGEshowed that production of the mutant GroELE129Xwith pBK was more efficient in DH10B E. coli,whereas expression with pSUP was better in BL21

Fig. 5. Expression of GroELE129X with different unnat-ural amino acid incorporation systems. Protein from 0.5 Lof culture was expressed, purified by anion exchange, andanalyzed by SDS-PAGE gel, as described in Materials andMethods. Lanes 2 and 3 correspond to expression with thepreviously reported pBK/pBADJY or pSUP/pTrc plas-mids, respectively. Lane 4 corresponds to expression withthe pEVOL/pTrc plasmid described here. Lanes 5 and 6are wild-type GroEL (GroELWT) expressed from the pTrcor pBAD plasmids, respectively. Lanes 2 and 6 correspondto expression in DH10B E. coli. Lanes 3, 4, and 5correspond to expression in BL21 E. coli, as described inSupplementary Fig. 2.


E. coli (Supplementary Fig. 2). However, pEVOLshowed similarly high levels of mutant proteinexpression regardless of E. coli strain, indicating thatthis system allows for greater versatility in E. coliexpression strains than existingmethods. Purificationof GroELE129X from 0.5-L cultures yielded 58 mg ofpurified full-length protein with pEVOL-pAcF, 24mgof purified full-length protein with pSUP-pAcF, and1.4 mg of purified full-length protein with pBK-pAcF(Fig. 5). For comparison, pTrc99a-GroELWT yielded72 mg of full-length GroEL, and pBAD-GroELWTyielded 26 mg of full-length GroEL. These trends inprotein yields are very similar to the results found inpET-GFPY151X assay (Fig. 2). Mass spectrometryanalysis of the GroELE129X protein confirmed unam-biguous incorporation of the pAcF unnatural aminoacid using the pBK, pSUP, or pEVOL system(Supplementary Fig. 3).

Application of pEVOL to other unnatural aminoacids

To simplify the use of aaRS mutants that incor-porate more than 30 unnatural amino acids intobacteria, we modified the pEVOL backbone toinclude NdeI-PstI and BglII-SalI unique restrictionsites that are compatible with the M. jannaschii aaRSgene. Alternatively, an overlap polymerase chainreaction (PCR) method was devised for one-stepsubcloning using BglII and PstI (see Materials andMethods for details). aaRS specific for p-azidophe-nylalanine (pAzF; azide, “click” chemistry, photo-crosslinker, IR probe),37 p-benzoylphenylalanine(pBpF; photocrosslinker),19 p-propargyloxyphenya-lanine (pPrF; alkyne, “click” chemistry),38 p-hydroxy-L-phenyllactic acid (PLA; backbone ester, tracelesstag),39 p-iodophenylalanine (pIF; heavy atom),23

p-cyanophenylalanine (pCNF; IR probe),22 p-carbo-

xymethylphenylalanine (pCmF; stable phospho-tyrosine mimic),40 L-3-(2-naphthyl)alanine (NapA;structural probe),41 p-boronophenylalanine (pBoFunique boronate reactivity),24 o-nitrophenylalanine(oNiF; backbone photolysis),42 (8-hydroxyquinolin-3-yl)alanine (HQA; metal ion binder),17 (2,2′-bipyridin-5-yl)alanine (BipyA; metal ion binder),43 tyrosine(wild type), and (7-hydroxycoumarin-4-yl)ethylgly-cine (CouA; fluorophore)18 were inserted into thepEVOL vector harboring one copy of tRNACUA

opt .Constructs were tested for their ability to insertthese unnatural amino acids in response to threeindependent amber mutations in the GFP gene(GFPK3X, GFPY151X, and GFPD133X) encoded inpET101 (Fig. 6a). The position of the TAG codonwas varied to eliminate context effects associatedwith the site of amber suppression. Protein expres-sion was performed in the presence or in theabsence of cognate amino acid (1 mM, except for0.2 mM pBpF) to demonstrate the fidelity ofunnatural amino acid incorporation with variousaaRS. Suppression with the unnatural amino acidpBpF was determined to be optimal at 0.2 mM,presumably due to slight toxicity at 1 mM.Unnatural amino acids were incorporated into

different sites in GFP with varied efficiencies, whichin general, depended on the site of incorporationmore than on the nature of the amino acid (Fig. 6a).Although it is clear that the protein context willaffect the incorporation of specific unnatural aminoacids differently, these results suggest that addi-tional factors influence the efficiency of unnaturalamino acid incorporation, such as the mRNAcontext of the UAG codon.26 Absolute rules forthese phenomena in E. coli are yet to be deciphered;for this reason, we typically test multiple candidatesites for incorporation efficiency with any givenunnatural amino acid. Of the aaRS tested in pEVOL,pEVOL-Tyr (wild-type tyrosyl-RS) demonstratedthe highest efficiency of amino acid incorporationwith an average yield (based on fluorescence) ofgreater than 50% that of GFPWT across the three GFPmutants (Fig. 6a). pEVOL-pAzF, pEVOL-pAcF,pEVOL-pIF, pEVOL-pCNF, and pEVOL-NapAwere also highly efficient at unnatural amino acidincorporation, with an average yield of greater than35% relative to GFPWT for all three mutants whenexpressed in the presence of their cognate unnaturalamino acid. All aaRS, except for PLA-RS, affordedan average fluorescence of 10%when expressed in thepresence of unnatural amino acid. Due to the labileester backbone of PLA, incorporation of this residueat certain positions may make it more susceptible tohydrolysis and subsequent loss of GFP fluorescence,as previously described.39 This is likely the reasonthat pEVOL-PLA/pET-GFPY151X failed to yield morethan 3% of the fluorescence of GFPWT (Fig. 6a).pEVOL-CouA could not be assayed by GFP fluores-cence in this assay due to interference from thecoumarin fluorophore; therefore, it was tested usingthe myoglobin assay described below (Fig. 6b).Several aaRS (pAzF, PLA, pIF, pCNF, pCmF, oNiF,

HQA, and pAcF) also led to background suppres-

Fig. 6. Unnatural amino acid incorporation as a function of aaRS. (a) Thirteen unnatural aaRS (described in the text)and wild-type tyrosyl-RS (Tyr) were tested for their ability to suppress three amber mutations in GFP [GFPK3X (GFP 3X),GFPY151X (GFP 151X), or GFPD133X (GFP 133X)]. Experiments were carried out in 2×YT-rich media in the presence (+) or inthe absence (−) of cognate amino acid. Fluorescence is shown relative to pEVOL-aaRS/pET-GFPWT. CouA-RS was notassayed due to interference from the coumarin fluorophore. Error bars, SD (n=3 for +pAcF; n=2 for −pAcF). (b) Myowithan amber mutant at position F107 (MyoF107X) was suppressed using pEVOL, with the aaRS listed above the gels. Proteinexpression was carried out in the presence (+; top) or in the absence (−; bottom) of cognate amino acid. CouA-RS, whichwas not present in (a), was assayed by myoglobin expression. Gels show affinity-purified 6×His-tagged myoglobin from10 ml of BL21(DE3) cultures analyzed by denaturing SDS-PAGE gel and stained with Coomassie brilliant blue. Forcomparison, the final lane in the top gel shows wild-type protein yields from pEVOL-aaRS/pET-MyoWT. MyoF107Xexpressions with pAzF are derived from a separate experiment.


sion of the GFPY151X amber mutant based onfluorescence intensity when expressed in the ab-sence of their cognate unnatural amino acid in 2×YT(rich) media. This is typically not observed inGMML (glycerol minimal medium+leucine), asdescribed in the primary references for the evolutionof these aaRS.17,19,22,23,31,37,39,40 Background incor-poration of this nature is typically caused byaminoacylation of the suppressor tRNACUA by theevolved aaRS with an endogenous amino acid(usually Phe or Tyr) in rich media in the absenceof its cognate unnatural amino acid. This back-ground incorporation is typically suppressed by theaddition of cognate unnatural amino acid to theexpression media. Therefore, to assess the degree ofbackground misaminoacylation occurring in thepresence of the cognate unnatural amino acidwhen expressed in rich media using the pEVOLsystem, we expressed MyoF107X in 2×YT media withall pEVOL-aaRS constructs and analyzed them withmass spectrometry (Fig. 6b). Again, full-lengthmyoglobin was detected for some aaRS when thecognate amino acid was withheld from the media, asdetermined by SDS-PAGE analysis (Fig. 6b, bot-

tom). For those aaRS that exhibited this backgroundsuppression activity in the absence of the unnaturalamino acid (pAzF, pBpF, PLA, pIF, pCNF, pCmF,oNiF, HQA, and pAcF) and for CouA-RS, affinity-purified MyoF107X expressed in the presence ofunnatural amino acid was analyzed by liquidchromatography mass spectrometry–electrosprayionization mass spectrometry. aaRS specific forpBpF, PLA, pIF, pCNF, pCmF, pAcF, and CouAproduced MyoF107X mutants with masses corres-ponding to the exclusive incorporation of theircognate unnatural amino acid (Supplementary Fig.3a–f, i, j). No other mass corresponding to one of thenative 20 amino acids at position 107 of myoglobinwas found by this liquid chromatography massspectrometry–electrospray ionization method,which is sensitive enough to detect contaminantprotein masses as low as 8% of the total protein. TheaaRS specific for oNiF, HQA, and pAzF showedsome background (25% tyrosine, 30% phenylala-nine, and 14% tyrosine/p-aminophenylalanine in-corporation into the TAG codon, respectively) inmyoglobin expressions carried out in rich media(see Supplementary Fig. 3g, h, k). This background


incorporation had previously been observed withoNiF-RS,42 but was not observed for HQA-RS orpAzF-RS in minimal media. For the isolated caseof pAzF, the mass spectra peaks identified as tyro-sine background are likely the result of themetabolic conversion of the pAzF amino acid top-aminophenylalanine.44,45 These results may indi-cate that a reevolution of these aaRS is warrantedor may indicate the continued use of minimalmedia for their expression.

Discussion

In an effort to optimize and standardize a bacterialexpression system for the incorporation of unnaturalamino acids into proteins, we have consolidated 14aaRS/tRNACUA pairs into a single highly adaptablevector system, pEVOL. Because overexpression offoreign aaRS and amber suppressor tRNAs in E. colican be toxic due to their interactions with theendogenous translation machinery [e.g., elongationfactor Tu (EF-Tu) or ribosomal components] and thesuppression of native TAG codons (which representapproximately 7% of native stop codons in E. coli),an inducible araBAD-aaRS gene was used to over-express the aaRS. An additional copy of the aaRSgene under the glnS′ promoter was found to furtherincrease protein yields and may aid in creating abasal level of aaRS that is available to the cell at thestart of induction. An inducible system for thetranscription of tRNACUA

opt was not required, sincebacteria expressing this suppressor tRNA exhi-bited normal growth rates. Although the cause oftRNACUA toxicity is not entirely known, evolutionof the optimized tRNACUA

opt was focused on theT-stem, which interacts with EF-Tu. Details of theevolution of tRNACUA

opt , as well as of evolvedsuppressor tRNAs specific for each unnaturalamino acid, are described elsewhere.32

The efficiency of unnatural amino acid incorpora-tion clearly depends on the site and the nature of theprotein, as demonstrated in the expression ofMyoS4X, MyoF107X, and MyoY152X (Fig. 2d) orGFPK3X, GFPY151X, and GFPD133X mutants with thepEVOL system (Fig. 6a). In addition to the effects oflocal protein structure on amino acid incorporationefficiency, mRNA context effects (which are thoughtto be, in large part, a result of competition betweenrelease factor 1 and the suppressor tRNACUA

25,26)likely play a significant role in the efficiency ofunnatural amino acid incorporation at any givensite. It should be noted that previous experimentsincorporating unnatural amino acids with pSUP,pBK, and pSUPAR3 vectors have afforded yields ofmutant proteins up to ∼90% of the yield of wild-type protein.27 However, since protein and nucleicacid context plays such an important role indetermining yields, it is not easy to directly compareexpression experiments from different sites ordifferent proteins. Therefore, to optimize ambersuppression with the pEVOL system, we intention-ally chose GFP and myoglobin mutants that

afforded less than 50% of wild-type protein. Indeed,yield improvements with the pEVOL series ofplasmids were relatively consistent across myoglo-bin mutants, as determined by SDS-PAGE analysis,and were roughly 250% greater than the previouslydeveloped pSUP system, ranging from roughly 35%to 50% of wild-type protein for most mutantaminoacyl-tRNA synthetase/tRNA pairs.The pEVOL vector is optimized for the general

incorporation of all unnatural amino acids that usemutantM. jannaschii tyrosyl-RS. We have attemptedto increase the overall yields of mutant proteinthrough expression in rich media under conditionswhere aminoacylation of tRNACUA by the unnaturalamino acid may be limiting. In doing so, we haveidentified a number of aaRS/tRNACUA pairs (e.g.,oNiF and HQA) with suboptimal catalytic efficien-cies and fidelities that can lead to diminished yieldsand decreased fidelities, which vary with the site ofincorporation. Background incorporation by theseaaRS/tRNACUA pairs in rich media can likely bereduced by further evolution of these aaRS (e.g.,increased stringency of the negative selectionimproved active-site libraries guided by X-raystructures of mutant aaRS,33,46–48 or selection in richmedia) or by increasing amino acid concentration.21

Alternatively, decreased fidelity can beminimized bythe use of minimal media49; however, this generallyresults in decreased yields of mutant proteins.Additional modifications to the expression system

may also lead to general increases in the yields ofmutant proteins or to improvements in the yields ofspecific mutants. For example, dipeptides have beenused to increase the solubility and cytoplasmicconcentrations of some unnatural amino acids suchas pBpF and may warrant exploration with otherunnatural amino acids.50 Increased amino acid con-centrations in the media may improve yields withsome unnatural amino acids, but can be a challengefor amino acids that are not commercially available.Generally, we have found that 1mMunnatural aminoacid in the growthmedia is sufficient.Modifications tothe host strain, such as mutation of EF-Tu andpeptidyl transferase or decreased expression of releasefactor 1 levels, are also expected to increase the yieldsof mutant proteins. The use of orthogonal ribosomeswould be expected to decrease any associated toxiceffect of such mutations.51

Amber suppression with pEVOL is not restrictedto the pET101 vector or to BL21(DE3) E. coli, nor arethe conditions reported here necessarily optimal forall proteins. However, these conditions representgood starting points for expression trials. Trendsfrom these experiments demonstrate that the effi-ciency of this incorporation system has littledependence on temperature or on the growthphase at which protein expression is induced, butrequires induction of the aaRS prior to or simulta-neous with target gene induction. GroELE129Xexpression with pTrc99a shows that pEVOL workswell for larger-scale protein expression and withother expression vectors and E. coli strains. Theseresults suggest that this new system will be com-


patible with a wide range of protein expressionprotocols and should afford improved yields ofmutant proteins containing unnatural amino acids.Furthermore, by consolidating all of the mutantaaRS into a single expression system, we have beenable to compare 14 unnatural aaRS under identicalconditions. This will benefit future applications byallowing greater predictability and reproducibilityfor the incorporation of distinct nonnatural aminoacids. Moreover, future improvements in method-ology can easily be incorporated into these vectorsand compared directly to the pEVOL system withthese GFP and myoglobin mutants. Current work isaimed at placing the more than 30 unnatural aaRSderived from M. jannaschii tyrosyl-RS into thepEVOL system. Pairs from other organisms suchas Methanosarcina mazei and Methanosarcina barkeriare also being tested with this new system.

Materials and Methods

Top10 or DH10B E. coli (Invitrogen) was used forcloning and amplifying plasmids. DNA was isolated fromE. coli using the PureLink Quick Plasmid Miniprep Kit(Invitrogen). Platinum pfx DNA polymerase (Invitrogen)was used for PCR. Restriction enzymes were purchasedfrom New England Biolabs, BL21(DE3) Star E. coli wasobtained from Invitrogen, and antibiotics were purchasedfrom Sigma-Aldrich or Fisher. The working antibioticconcentrations were as follows: chloramphenicol, 50 μg/ml; ampicillin, 100 μg/ml; tetracycline, 25 μg/ml; kana-mycin, 50 μg/ml. All cultures were grown in 2×YT (Difco)media. All assay plates were black clear-bottom200 μl×96-well assay plates (Costar). Constructed plas-mids were confirmed by sequencing. Primers weresynthesized by Integrated DNA Technology (San Diego),and their sequences are listed in Supplementary Table 1.Unnatural amino acids were obtained or synthesized fromthe sources or schemes indicated in their primarypublications. Plasmid maps were constructed usingVector NTI Advance 11 (Invitrogen).

Construction of the pEVOL series of vectors

The p15A origin, chloramphenicol acetyltransferase(CmR) gene, proK promoter and terminator, and araCgene were derived from pSUPAR321 or its predecessorpSUP.27 To create the glnS′-pAcF-RS version of pEVOL,we PCR amplified pSUPAR3-pAcF-RS (from B. Geier-stanger) with the primers glnSF and glnSR to exclude thearaBAD-aaRS-rrnB and araC coding regions. The primersintroduced an AatII restriction site to which the ends of thevector were ligated back together to create pEVOL-a. Tocreate the araBAD-aaRS-rrnB version of pEVOL-b, we PCRamplified pSUPAR3 with the primers PstI-proK F andNdeI-araBAD R to exclude the glnS′-aaRS coding region.The pAcF-RS gene was separately amplified with theprimers aaRS F (NdeI) 2 and aaRS F (PstI) FIX from pBK-pAcF.31 Both PCR products were digested with PstI andNdeI and ligated to create pEVOL-b.Suppressor tRNA regions for pEVOL-c, pEVOL-d, and

pEVOL-e were constructed as follows. The tricistronictRNACUA

Tyr gene cluster was derived from pSUPAR3 andis further described by Ryu and Schultz.27 A bicistronictRNACUA

Tyr cluster was serendipitously achieved by

spontaneous homologous recombination of the tricistro-nic gene sequence during cloning. The monocistronictRNACUA

Tyr was PCR amplified from a pSUPAR constructharboring one copy of tRNACUA

Tyr (from B. Geierstanger).All tRNA regions were PCR amplified with the primerstRNA_1_F and tRNA_3_R, digested with XhoI andApaLI, and ligated into the similarly digested vectorbackbone to create pEVOL-c, pEVOL-d, and pEVOL-e.To create the tricistronic tRNACUA

opt gene cluster inpEVOL-g, we followed the protocol of Ryu and Schultz,using the primers tRNA_1_F and tRNA_1_R (set 1); tRNA_2_F New and tRNA_2_R (set 2); and tRNA_3_F New andtRNA_3_R (set 3), to insert the valU-valX and ileT-alaTlinker sequences between three copies of tRNACUA

opt .27 Theend product was reamplified with the primers tRNA_1_Fand tRNA_3_R, digested with XhoI and ApaLI, andligated into the similarly digested vector backbone. Themonocistronic tRNACUA

opt gene, including part of the proKpromoter (to the ApaLI site) and the proK terminator (tothe XhoI site), was synthesized by DNA 2.0 (Menlo Park,CA), PCR amplified with the primers tRNA_1_F andtRNA_3_R, digested with XhoI and ApaLI, and ligatedinto the similarly digested vector to create pEVOL-f.To create the dual glnS′/araBAD version of pEVOL, we

used a modified method from Cellitti et al.21 The pAcF-RSgene was PCR amplified from pBK-pAcF6 with theprimers aaRS NcoI F and aaRS SalI R, digested withNcoI and SalI, and ligated into a pBAD vector (Invitrogen)to create pBAD-pAcF. The region corresponding toaraBAD-aaRS-rrnB was PCR amplified from pBAD-pAcFwith the primers aaRS pBADF and aaRS pBad R. PrimeraaRS pBADF extended from the BamHI site in the araBADpromoter to the start of the aaRS gene and was used toinsert a BglII site just 5′ of the aaRS. The PCR product wasdigested with BamHI and SacI, and ligated into thesimilarly digested pSUPAR3 (already harboring anotherpAcF-RS gene under the glnS′ promoter). From thisconstruct, the tRNA region could be modified using theaforementioned ApaLI and XhoI sites. Future subcloningof the aaRS gene to both the glnS′ region and the araBADregion of pEVOL could use the uniqueNdeI-PstI and BglII-SalI sites and did not need to be cloned into pBAD first.An overlap PCR method was devised to insert two

copies of any evolved M. jannaschii tyrosyl-RS intopEVOL. Briefly, the aaRS was PCR amplified in twoseparate amplifications with the primers aaRS pBAD F–aaRS1 R (copy 1) and aaRS2 F–aaRS2 R (copy 2). The DNAregion corresponding to the rrnB terminator and the glnS′promoter was PCR amplified from pSUPAR3 or pEVOLwith the primers rrnB F and rrnB R (rrnB region). The rrnBregion was individually ligated to copies 1 and 2 byoverlap PCR with the primers aaRS pBAD F–rrnB R(overlap 1) and rrnB F–aaRS2 R (overlap 2), respectively.Overlap PCR products were then ligated by anotheroverlap PCR with the primers BglII dia and PCR4,digested with BglII and PstI, and ligated into the similarlydigested pEVOL plasmid. All intermediate steps werepurified by agarose gel separation to prevent thecarryover of parent vectors or unwanted products. Toadd the D286R mutation to both copies of the aaRS, wereplaced aaRS1 R and aaRS2 R with the primers aaRS1D-R R and aaRS2 D-R R, respectively.

Construction of pET vectors

GFP(uv) was derived from pLeiG, as described byWang et al.34 Mutant GFPK3X was created by PCR with theprimers GFP K3 F and GFP R. Mutant GFPD133X was


created by overlap PCR with the primers GFP D133 F–GFP R (set 1) and GFP D133 R–GFP F (set 2). The GFPY151Xmutant was a standard clone in the Schultz laboratory.Myo with a C-terminal 6×His tag was derived frompBAD/JYAMB-4TAG-Myo.18 Myoglobin mutant MyoS4Xwas a standard clone in the Schultz laboratory. MutantMyoF107X was created by overlap PCR with the primersMyo F107 F–Myo R (set 1) and Myo F107 R–Myo F (set 2).Mutant MyoY152X was created by PCR with the primersMyo Y152 R and Myo F. All GFP and Myo mutants orwild-type GFP or Myo were amplified by the primer GFPF–GFP R or Myo F–Myo R, respectively, and was “TOPO-cloned” into pET101D following protocols from theChampion pET Directional TOPO Expression Kit (cat noK100-01; Invitrogen).

GFP expression protocol

pEVOL constructs with varied promoters or tRNAregions (pEVOL-a–pEVOL-g, pEVOL-Tyr, or pEVOL), orvaried aaRS genes were cotransformed with pET101-GFPmutants into BL21(DE3) Star E. coli. As a control, pET101-GFPWT was similarly cotransformed into BL21(DE3) withpEVOL-aaRS, so cultures could be grown under identicalconditions. A pET101 vector encoding a GFP gene insertedbackwards (nonfunctional) was cotransformed into BL21(DE3) with pEVOL to serve as background control. Asingle colony from each cotransformation was grownovernight at 37 °C in 1 ml of 2×YTmedia in a 2ml×96-wellblock supplemented with ampicillin and chlorampheni-col. Overnight cultures were diluted to an OD600 of 0.2(1 ml of culture in 96-well blocks) and grown forapproximately 1.5 h to OD600=0.6–0.8 before induction.Cultures were split into 5×180 μl in 96-well assay platesand induced by adding a 10% culture volume (20 μl) of2×YT media containing 10 mM unnatural amino acid(unless no unnatural amino acid is indicated), 0.2%arabinose, and 10 mM IPTG. Growth was continued at30 °C for 14–16 h before the cultures were cooled to roomtemperature for 30 min. GFP fluorescence was measuredin 96-well assay plates with a SpectraMAX GeminiEMplate reader (Molecular Devices), with excitation at395 nm, emission at 510 nm, and cutoff at 495 nm.Fluorescence readings from each well were normalized toOD600 by reading at 600 nm with a SpectraMAX 250 platereader (Molecular Devices). Background fluorescence(measured from pET101 encoding a nonfunctional GFP)was subtracted from the readings, and fluorescence wasreported as a percentage of pEVOL-aaRS/pET-GFPWT.

Myoglobin expression protocol

Transformation and protein expression were carried outsimilarly to GFP expression experiments. As control,pET101-MyoWT was cotransformed into BL21(DE3) withpEVOL-aaRS, so cultures could be grown under identicalconditions. Single colonies from pEVOL/pET-Myocotransformations were grown overnight at 37 °C tosaturation in 5 ml of 2×YT media in 14-ml Falcon tubes(Fisher) supplemented with ampicillin and chloramphen-icol. Overnight cultures were diluted to an OD600 of 0.2(20 ml of culture in 50-ml Corning tubes) and grown forapproximately 1.5 h to OD600=0.6–0.8 before induction.Cultures were split into 2×9 ml in 14-ml Falcon tubes andinduced by adding a 10% culture volume (1 ml) of 2×YTmedia containing 10 mM unnatural amino acid (1 ml of2×YT media lacking unnatural amino acid was added toone of the 9-ml cultures), 0.2% arabinose, and 10 mM

IPTG. After expression for 14–16 h at 37 °C, 10-mlcultures were pelleted at 6000 relative centrifugal force(rcf) and frozen at −80 °C. Pellets were thawed andlysed for 30 min with Bugbuster (Novagen) supplemen-ted with ethylenediaminetetraacetic-acid-free Completeprotease inhibitor cocktail (Roche), 0.5 mg/ml lysozyme(Sigma), and 2.5 U/ml benzonase (Novagen). Lysateswere centrifuged at 35,000 rcf, and myoglobin (with a6×His at the C-terminus) was purified with a Ni-NTAspin column (Qiagen). Columns were washed twice with600 μl of native wash buffer [50 mM NaH2PO4, 300 mMNaCl, and 20 mM imidazole (pH 8.0)] and eluted twicewith 50 μl of native elution buffer (wash buffer plus230 mM imidazole). Eluted samples were analyzed on aTris–Gly SDS-PAGE gel (Invitrogen) and stained withCoomassie brilliant blue. Unnatural amino acid incor-poration was confirmed by mass spectrometry and islisted in Supplementary Fig. 3.

Expression of pBK-pAcF/pLeiGY151X andpSUP-pAcF/pET-GFPY151X

pBK-pAcF had been previously described by Wang etal.,31 and pLei had been previously described by Wang etal.34 To create pLeiGY151X and pLeiGWT, we PCR amplifiedGFPY151X or GFPWT, respectively, with the primers GFP FNdeI and GFP R SacI, digested with NdeI and SacI, andligated into the similarly digested pLei vector. pBK-pAcFwas cotransformed with pLeiGY151X or pLeiGWT intoDH10B E. coli. pSUP-pAcF had been previously describedby Ryu and Schultz.27 The vector was cotransformed withpET-GFPY151X or pET-GFPWT intoBL21(DE3) for expressionexperiments. Protein expression for both pSUP and pBKwas carried out as described in theGFP expression protocol,except that arabinose was withheld from the media.

E. coli DH10B and BL21(DE3) growth curves

pEVOL-pAcF with one copy of tRNACUAopt , or pEVOL-

pAcF with one copy of tRNACUATyr was transformed into

DH10B or BL21(DE3) E. coli. As a control, a 400-bp regionof DNA was PCR amplified from a noncoding region ofpET101 (randomly chosen) by the PCR primers ApaLI Fand XhoI R, digested with ApaLI and XhoI, and ligatedinto the corresponding sites in pEVOL between the proKpromoter and the proK terminator. One colony from eachtransformationwas grown overnight to saturation in 2×YTmedia supplemented with 50 μg/ml chloramphenicol.Cultures were diluted to an OD600 of 0.05 and aliquotedinto 96-well assay plates in quadruplicate. Plates weregrown in a SpectraMAX 250 plate reader (MolecularDevices) and read (at 600 nm) every 3 min with mixing at37 °C for 8 h. Specific growth rates (μ) were determinedwhen the cells appeared to be in maximum growth(OD600=0.2–0.7 for 200 μl of culture). Rates were calculat-ed by the time it took for cells to go from OD1 set to 0.2 toOD2 set to 0.7, using the following formula (t in hours):

A =ln

OD1

OD2

� �

t2 � t1

Optimization of induction parameters

pEVOL-pAcF/pET-GFPY151X or pEVOL-pAcF/pET-GFPWT cotransformed in BL21(DE3) E. coli was used for


all induction assays. A starter culture was picked from asingle colony and grown overnight to saturation at 37 °Cin 2×YT media supplemented with chloramphenicol andampicillin. Cultures were diluted to an OD600 of 0.2 andgrown to OD600=0.5–0.7 before being aliquoted in trip-licate into 96-well assay plates with varied concentrationsof inducers (0.0002–2% arabinose or 0.01–100 mM IPTG ornone) and pAcF amino acid, where necessary. Eachcondition was replicated in three wells. Growth wascontinued for 14–16 h at 30 °C, and fluorescence wasassayed as described above.To assay induction order,we diluted starter cultures to an

OD600 of 0.05, and 180 μl was aliquoted into 96-well assaysplates in quadruplicate for each condition.Cellswere grownat 30 °Cwith shaking in a SpectraMAX 250 plate reader andmonitored at 600 nm. A factor of 3.95 (after blanking) wasdetermined for the conversion of the absorbance at 600 nmfor a 180 μl culture in an assay plate into a standard 1-cmOD600. Cultures were induced as described with 20 μl of2×YT media. After the final induction, the cultures weremoved to a 30 °C shaking incubator for 14–16 h, and GFPfluorescence was reported as described above.The effect of temperature was assayed by diluting

starter cultures to an OD600 of 0.15 and growing themto an OD600 of 0.4. Cultures were then split and eithercooled to 25 °C, cooled to 30 °C, or kept at 37 °C inseparate shaking incubators. Growth was continued untilOD600=0.6–0.8, inducers and pAcF amino acid wereadded as previously described, and expression continuedfor 14–16 h at the indicated temperatures. GFP fluores-cence was reported as described above.

Expression of GroEL

All GroEL constructs were obtained from Eli Chapman.BL21 E. coli used for the expression of GroEL did not havea DE3 prophage. pBK-pAcF was cotransformed withpBADJY-GroELE129X into BL21 or DH10B E. coli andcultured with kanamycin and tetracycline. pBAD-GroEL-WT or pTrc99a-GroELWT was individually transformedinto DH10B or BL21 and grown with ampicillin. pTrc99a-GroELE129X could not be cotransformed with pEVOL orpSUP for unknown reasons; therefore, pTrc99a-GroEL E129Xwas transformed into BL21 or DH10B E. coli, then a singlecolony was picked for each, cultured with ampicillin, andmade competent. Competent DH10B or BL21 E. coliharboring pTrc99a-GroELE129X was transformed withpEVOL-pAcF or pSUP-pAcF and cultured with chloram-phenicol and ampicillin. Single colonies for pBK-pAcF/pBADJY-GroELE129X, pSUP-pAcF/pTrc99a-GroELE129X,pEVOL-pAcF/pTrc99a-GroELE129X, pTrc99a-GroELWT, orpBAD-GroELWT in DH10B or BL21 E. coli (10 total) werepicked into 5 ml of 2×YT media, and test expressions werecarried out identically to myoglobin expressions, exceptthat no protein was purified. Crude cell lysates wereanalyzed after 10 h and 20 h as follows: 7 μl of culturewas diluted with 23 μl of water and 15 μl of SDS loadingbuffer and boiled for 10 min, and 10 μl of the crude celllysate was run on an SDS-PAGE gel and stained withCoomassie brilliant blue (Supplementary Fig. 2). Proteinyields did not appear significantly different between 10 hand 20 h of expression.For 0.5-L expression experiments, pBK-pAcF/pBADJY-

GroELE129X and pBAD-GroELWT were transformed intoDH10B, and pSUP-pAcF/pTrc99a-GroELE129X, pEVOL-pAcF/pTrc99a-GroELE129X, and pTrc99a-GroELWT weretransformed into BL21. Single colonies were picked into30 ml of 2×YT media supplemented with appropriate

antibiotics and grown overnight to saturation at 37 °C.Overnight cultures were diluted to an OD600 of 0.1 in500 ml of 2×YT media supplemented with appropriateantibiotics in 2-L nonbaffled shake flasks. Cultures weregrown for approximately 3 h at 37 °C (2.5 h for pBAD-GroELWT or pTrc99a-GroELWT) to an OD600 of 1.0 beforeinduction by addition of 10 ml of 2×YT media containing50 mM pAcF amino acid (except for GroELWT expressions,where 10 ml of 2×YT media without pAcF was added),0.5 ml of 20% arabinose (except for pTrc99-GroELWTculture), and 0.5 ml of 1 M IPTG (only cultures withpTrc99a plasmids). After the cells had been shaken at250 rpm for 14 h at 37 °C, they were pelleted at 6000 rcf.Samples were maintained at 4 °C throughout purification.The supernatant was removed, and the pellets wereresuspended in 10 ml of breaking buffer [50 mM Tris(pH 7.4) and 1 mMDTT] and lysed with a Micro Fluidizer(Micro Fluidics Corp.). Lysates were cleared by centrifu-gation at 100,000 rcf and loaded onto a 150-ml FFQ anion-exchange column (Pharmacia) equilibrated with breakingbuffer. The columnwas washed with 3 column volumes of0.2 M NaCl in breaking buffer and eluted with a gradientto 0.5 M NaCl in breaking buffer over 12 column volumesusing an Atka purifier (Pharmacia). Fractions thatcontained GroEL were identified by Coomassie-stainedSDS-PAGE, pooled, and quantified by a bicinchoninic acidassay (Pierce) with bovine serum albumin standard andshown in Fig. 5. Protein masses demonstrating incorpo-ration of pAcF are listed in Supplementary Fig. 3.

Expression of pEVOL with multiple aaRS

For GFP expression, single colonies from distinctpEVOL-aaRS cotransformed with pET-GFP mutantswere picked into 1 ml of 2×YT media supplementedwith chloramphenicol and ampicillin in a 2-ml 96-wellblock (Nunc). pEVOL/pET-GFPWT and pEVOL/pET-blank were also included as controls. Cultures weregrown to saturation overnight at 37 °C, and OD600 wasmeasured by removing 200 μl of culture onto a 96-wellassay plate and reading with a SpectraMAX 250 platereader. OD600 was converted into a 1-cm OD600 using theaforementioned 3.95 conversion factor. Cultures werediluted evenly to an OD600 of 0.25 in a 2-ml 96-well block,and growth continued at 37 °C. After 2 h, 180 μl of culturewas aliquoted into 3×96-well assay plates containingappropriate unnatural amino acids and inducers (as notedin previous GFP expressions) or 2×96-well assay plateslacking any unnatural amino acid but containing inducers.Growth was continued at 30 °C for 14–16 h, and GFPfluorescence was reported as described above.

Acknowledgements

T.S.Y. is grateful for an Achievement Rewards forCollege Scientists Scholarship. We thank B. Geier-stanger and S. Cellitti for the pSUPAR3 vector andvaluable suggestions; C. Melancon and J. Guo forthe evolution of novel tRNAs; E. Brustad, C.Dilworth, and with J. McBride for their help withthe organization of this methodology; and E.Chapman for his help with GroEL expression andpurification. This work was funded by grant DE-FG03-00ER46051 from the Division of Materials


Sciences, Department of Energy. This is manuscript20213 of the Scripps Research Institute.

Supplementary Data

Supplementary data associated with this articlecan be found, in the online version, at doi:10.1016/j.jmb.2009.10.030

References

1. Xie, J. & Schultz, P. G. (2006). A chemical toolkit forproteins—an expanded genetic code. Nat. Rev. Mol.Cell Biol. 7, 775–782.

2. Wang, L., Xie, J. & Schultz, P. G. (2006). Expanding thegenetic code. Annu. Rev. Biophys. Biomol. Struct. 35,225–249.

3. Xie, J. & Schultz, P. G. (2005). Adding amino acids tothe genetic repertoire. Curr. Opin. Chem. Biol. 9,548–554.

4. Wang, L. & Schultz, P. G. (2004). Expanding thegenetic code. Angew. Chem. Int. Ed. Engl. 44, 34–66.

5. England, P. M. (2004). Unnatural amino acid muta-genesis: a precise tool for probing protein structureand function. Biochemistry, 43, 11623–11629.

6. Zhang, Z. W., Smith, B. A. C., Wang, L., Brock, A.,Cho, C. & Schultz, P. G. (2003). A new strategy forthe site-specific modification of proteins in vivo.Biochemistry, 42, 6735–6746.

7. Chen, S., Schultz, P. G. & Brock, A. (2007). Animproved system for the generation and analysis ofmutant proteins containing unnatural amino acids inSaccharomyces cerevisiae. J. Mol. Biol. 371, 112–122.

8. Young, T. S., Ahmad, I., Brock, A. & Schultz, P. G.(2009). Expanding the genetic repertoire of themethylotrophic yeast Pichia pastoris. Biochemistry, 48,2643–2653.

9. Liu, W., Brock, A., Chen, S. & Schultz, P. G. (2007).Genetic incorporation of unnatural amino acids intoproteins in mammalian cells. Nat. Methods, 4, 239–244.

10. Sakamoto, K., Hayashi, A., Sakamoto, A., Kiga, D.,Nakayama, H., Soma, A. et al. (2002). Site-specificincorporation of an unnatural amino acid intoproteins in mammalian cells. Nucleic Acids Res. 30,4692–4699.

11. Wang, W., Takimoto, J. K., Louie, G. V., Baiga, T. J.,Noel, J. P., Lee, K. F. et al. (2007). Genetically encodingunnatural amino acids for cellular and neuronalstudies. Nat. Neurosci. 10, 1063–1072.

12. Anderson, J. C., Wu, N., Santoro, S. W., Lakshman, V.,King, D. S. & Schultz, P. G. (2004). An expandedgenetic code with a functional quadruplet codon. Proc.Natl Acad. Sci. USA, 101, 7566–7571.

13. Wang, Q., Parrish, A. R. &Wang, L. (2009). Expandingthe genetic code for biological studies. Chem. Biol. 16,323–336.

14. Chen, P. R., Groff, D., Guo, J., Ou, W., Cellitti, S.,Geierstanger, B. H. & Schultz, P. G. (2009). A facilesystem for encoding unnatural amino acids in mam-malian cells. Angew. Chem. Int. Ed. Engl. 48, 4052–4055.

15. Wang, L., Magliery, T. J., Liu, D. R. & Schultz, P. G.(2000). A new functional suppressor tRNA/aminoacyl-tRNA synthetase pair for the in vivo incorporation ofunnatural amino acids into proteins. J. Am. Chem. Soc.122, 5010–5011.

16. Wang, L. & Schultz, P. G. (2001). A general approach

for the generation of orthogonal tRNAs. Chem. Biol. 8,883–890.

17. Lee, H. S., Spraggon, G., Schultz, P. G. & Wang, F.(2009). Genetic incorporation of a metal-ion chelatingamino acid into proteins as a biophysical probe. J. Am.Chem. Soc. 131, 2481–2483.

18. Wang, J., Xie, J. & Schultz, P. G. (2006). A geneticallyencoded fluorescent amino acid. J. Am. Chem. Soc. 128,8738–8739.

19. Chin, J. W., Martin, A. B., King, D. S., Wang, L. &Schultz, P. G. (2002). Addition of a photocrosslinkingamino acid to the genetic code of Escherichia coli. Proc.Natl Acad. Sci. USA, 99, 11020–11024.

20. Deiters, A., Geierstanger, B. H. & Schultz, P. G. (2005).Site-specific in vivo labeling of proteins for NMRstudies. ChemBioChem, 6, 55–58.

21. Cellitti, S. E., Jones, D. H., Lagpacan, L., Hao, X.,Zhang, Q., Hu, H. et al. (2008). In vivo incorporation ofunnatural amino acids to probe structure, dynamics,and ligand binding in a large protein by nuclearmagnetic resonance spectroscopy. J. Am. Chem. Soc.130, 9268–9281.

22. Schultz, K. C., Supekova, L., Ryu, Y. H., Xie, J. M.,Perera, R. & Schultz, P. G. (2006). A genetically encodedinfrared probe. J. Am. Chem. Soc. 128, 13984–13985.

23. Xie, J. M., Wang, L., Wu, N., Brock, A., Spraggon, G. &Schultz, P. G. (2004). The site-specific incorporation ofp-iodo-L-phenylalanine into proteins for structuredetermination. Nat. Biotechnol. 22, 1297–1301.

24. Brustad, E., Bushey, M. L., Lee, J. W., Groff, D., Liu,W.& Schultz, P. G. (2008). A genetically encodedboronate-containing amino acid. Angew. Chem. Int.Ed. Engl. 47, 8220–8223.

25. Mottagui-Tabar, S. & Isaksson, L. A. (1997). Only thelast amino acids in the nascent peptide influencetranslation termination in Escherichia coli genes. FEBSLett. 414, 165–170.

26. Short, G. F., III, Golovine, S. Y. & Hecht, S. M. (1999).Effects of release factor 1 on in vitro protein translationand the elaboration of proteins containing unnaturalamino acids. Biochemistry, 38, 8808–8819.

27. Ryu, Y. & Schultz, P. G. (2006). Efficient incorporationof unnatural amino acids into proteins in Escherichiacoli. Nat. Methods, 3, 263–265.

28. Chang, A. C. Y. & Cohen, S. N. (1978). Constructionand characterization of amplifiable multicopy DNAcloning vehicles derived from p15a cryptic mini-plasmid. J. Bacteriol. 134, 1141–1156.

29. Plumbridge, J. & Soll, D. (1987). The effect of dammethylation on the expression of glnS in Escherichiacoli. Biochimie, 69, 539–541.

30. Guzman, L. M., Belin, D., Carson, M. J. & Beckwith, J.(1995). Tight regulation, modulation, and high-levelexpression by vectors containing the arabinose p-badpromoter. J. Bacteriol. 177, 4121–4130.

31. Wang, L., Zhang, Z. W., Brock, A. & Schultz, P. G.(2003). Addition of the keto functional group to thegenetic code of Escherichia coli. Proc. Natl Acad. Sci.USA, 100, 56–61.

32. Guo, J., Melancon, C. E., III, Lee, H. S., Groff, D. &Schultz, P. G. (submitted). Evolution of ambersuppressor tRNAs for efficient bacterial productionof unnatural amino acid-containing proteins. Angew.Chem. Int. Ed. Engl.

33. Kobayashi, T., Nureki, O., Ishitani, R., Yaremchuk, A.,Tukalo, M., Cusack, S. et al. (2003). Structural basis fororthogonal tRNA specificities of tyrosyl-tRNA synthe-tases for genetic code expansion. Nat. Struct. Biol. 10,425–432.

http://dx.doi.org/10.1016/j.jmb.2009.10.030http://dx.doi.org/10.1016/j.jmb.2009.10.030


34. Wang, L., Xie, J., Deniz, A. A. & Schultz, P. G. (2003).Unnatural amino acid mutagenesis of green fluores-cent protein. J. Org. Chem. 68, 174–176.

35. Siegele, D. A. & Hu, J. C. (1997). Gene expression fromplasmids containing the araBAD promoter at sub-saturating inducer concentrations represents mixedpopulations. Proc. Natl Acad. Sci. USA, 94, 8168–8172.

36. Braig, K., Otwinowski, Z., Hegde, R., Boisvert, D. C.,Joachimiak, A., Horwich, A. L. & Sigler, P. B. (1994).The crystal structure of the bacterial chaperoninGroEL at 2.8 Å. Nature, 371, 578–586.

37. Chin, J. W., Santoro, S. W., Martin, A. B., King, D. S.,Wang, L. & Schultz, P. G. (2002). Addition of p-azido-L-phenylalanine to the genetic code of Escherichia coli.J. Am. Chem. Soc. 124, 9026–9027.

38. Deiters, A. & Schultz, P. G. (2005). In vivo incorpora-tion of an alkyne into proteins in Escherichia coli.Bioorg. Med. Chem. Lett. 15, 1521–1524.

39. Guo, J. T., Wang, J. Y., Anderson, J. C. & Schultz, P. G.(2008). Addition of an alpha-hydroxy acid to the geneticcode of bacteria. Angew. Chem. Int. Ed. 47, 722–725.

40. Xie, J. M., Supekova, L. & Schultz, P. G. (2007). Agenetically encoded metabolically stable analogue ofphosphotyrosine in Escherichia coli. ACS Chem. Biol. 2,474–478.

41. Wang, L., Brock, A. & Schultz, P. G. (2002). Adding L-3-(2-naphthyl)alanine to the genetic code of E. coli.J. Am. Chem. Soc. 124, 1836–1837.

42. Peters, F. B., Brock, A., Wang, J. Y. & Schultz, P. G.(2009). Photocleavage of the polypeptide backbone by2-nitrophenylalanine. Chem. Biol. 16, 148–152.

43. Xie, J. M., Liu, W. S. & Schultz, P. G. (2007). Agenetically encoded bidentate, metal-binding aminoacid. Angew. Chem. Int. Ed. 46, 9239–9242.

44. Leyva, E., Platz, M. S., Persy, G. & Wirz, J. (2002).Photochemistry of phenyl azide: the role of singlet andtriplet phenylnitrene as transient intermediates. J. Am.Chem. Soc. 108, 3783–3790.

45. Schrock, A. K. & Schuster, G. B. (2002). Photochemistryof phenyl azide: chemical properties of the transientintermediates. J. Am. Chem. Soc. 106, 5228–5234.

46. Liu, W., Alfonta, L., Mack, A. V. & Schultz, P. G.(2007). Structural basis for the recognition of para-benzoyl-L-phenylalanine by evolved aminoacyl-tRNA synthetases. Angew. Chem. Int. Ed. Engl. 46,6073–6075.

47. Turner, J. M., Graziano, J., Spraggon, G. & Schultz,P. G. (2006). Structural plasticity of an aminoacyl-tRNAsynthetase active site. Proc. Natl Acad. Sci. USA, 103,6483–6488.

48. Zhang, Y., Wang, L., Schultz, P. G. & Wilson, I. A.(2005). Crystal structures of apo wild-type M.jannaschii tyrosyl-tRNA synthetase (TyrRS) and anengineered TyrRS specific for O-methyl-L-tyrosine.Protein Sci. 14, 1340–1349.

49. Farrelli, I. S., Toroney, R., Hazen, J. L., Mehl, R. A. &Chin, J. W. (2005). Photo-cross-linking interactingproteins with a genetically encoded benzophenone.Nat. Methods, 2, 377–384.

50. Huang, L.-Y., Umanah, G., Hauser, M., Son, C.,Arshava, B., Naider, F. & Becker, J. M. (2008).Unnatural amino acid replacement in a yeast Gprotein-coupled receptor in its native environment.Biochemistry, 47, 5638–5648.

51. Wang, K., Neumann, H., Peak-Chew, S. Y. & Chin,J. W. (2007). Evolved orthogonal ribosomes enhancethe efficiency of synthetic genetic code expansion.Nat. Biotechnol. 25, 770–777.

An Enhanced System for Unnatural Amino Acid Mutagenesis in E. coliIntroductionResultsGeneral construction of pEVOLOptimization of tRNA and aaRS copy numberEffects of tRNA and aaRS mutationsComparison of pEVOL with other expression �systemsEffects of pEVOL on E. coli growth ratesOptimization of mutant protein expression �conditionsExpression of a model protein (GroEL) under �optimized conditionsApplication of pEVOL to other unnatural amino �acids

DiscussionMaterials and MethodsConstruction of the pEVOL series of vectorsConstruction of pET vectorsGFP expression protocolMyoglobin expression protocolExpression of pBK-pAcF/pLeiGY151X and �pSUP-pAcF/pET-GFPY151XE. coli DH10B and BL21(DE3) growth curvesOptimization of induction parametersExpression of GroELExpression of pEVOL with multiple aaRS

AcknowledgementsSupplementary DataReferences

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