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Protein Expression and Purification 15, 115–120 (1999)Article ID prep.1998.0999, available online at http://www.idealibrary.com on

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igh-Level Expression and Single-Step Purification ofeucyl-tRNA Synthetase from Escherichia coli

ianfeng Chen, Yong Li, Enduo Wang,1 and Yinglai Wangtate Key Laboratory of Molecular Biology, Shanghai Institute of Biochemistry,cademia Sinica, Shanghai, 200031, China

eceived July 27, 1998, and in revised form September 21, 1998

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A T7 promoter-based His6-tagging vector has beenonstructed that directs the synthesis in Escherichiaoli of fusion proteins containing a stretch of six his-idine residues at the N terminus. The vector allowsverproduction and single-step purification of His6-usion protein by immobilized metal (Ni21) chelate af-nity chromatography. The gene encoding leucyl-RNA synthetase (leuS) was cloned into this vectornd expressed in high level. The specific activity of theynthetase in the crude extract of E. coli JM109(DE3)ransformant containing the His6-tagging vector withhe gene leuS was approximately 110 times that ofM109(DE3) (the host strain without the vector). Theverproduced His6-fusion leucyl-tRNA synthetase cane purified to homogeneity under native conditionsithin 2 h by one-step affinity chromatography withn overall yield of 55%. The His6-tag at the N terminusf leucyl-tRNA synthetase did not affect its aminoacy-ation activity or the secondary structure. © 1999

cademic Press

Aminoacyl-tRNA synthetases arose early in evolu-ion and are believed to be a group of ancient enzymes1) which catalyze the reaction for precisely chargingRNAs with their cognate amino acids. Based on theonserved amino acid sequences and crystal struc-ures, these enzymes are divided into two majorlasses, class I and II, each with characteristic se-uence and structural motifs, which form the substrateinding and catalytic sites (2–5). The 10 class I en-ymes share HIGH and KMSKS motifs and containctive sites based on the Rossmann fold (a parallel-sheet nucleotide-binding fold). The two motifs form

1 To whom correspondence should be addressed at Shanghai Insti-ute of Biochemistry, Academia Sinica, 320 Yue-Yang Road, Shang-ai 200031, P. R. China. Fax: 10086-21-64338357. E-mail: wed@

erver.shcnc.ac.cn. p

046-5928/99 $30.00opyright © 1999 by Academic Pressll rights of reproduction in any form reserved.

art of the ATP-binding site (6–8). The 10 class IIynthetases have different conserved sequence motifsalled motifs 1, 2, and 3 and the antiparallel b-sheeturrounded by a-helices form their active sites (4,9).Escherichia coli leucyl-tRNA synthetase (LeuRS),2

hich belongs to class I, is a single-chain polypeptide of60 amino acid residues, with molecular weight ofbout 97.3 kDa as deduced from the gene leuS (10,11).he gene encoding E. coli LeuRS has been obtained

rom l15D7 clone of E. coli genomic library by comple-entation of a LeuRS temperature-sensitive mutantL231 in our lab (11). Some properties of this syn-

hetase were studied (12). In order to have deep insightnto the relationships between the structure and func-ion of the synthetase, crystallization of this enzyme isecessary. So far, the crystal structures of 16 of 20minoacyl-tRNA synthetases have been presented andeuRS was one of the 4 synthetases without crystaltructures (13). However, the expression of LeuRS in. coli transformant containing leuS was relatively lownd the purification procedure was complex (12), whichade it very difficult to obtain some of its mutants

ecause of their instability, let alone the production ofarge amounts of the purified enzyme needed for itsrystallization. A new system for overproduction ofeuRS and a simple, efficient, and fast method for theurification of this enzyme was required. Here, weeport the overproduction and purification of LeuRS byingle-step immobilized metal (Ni21) affinity chroma-ography procedure (14,15).

ATERIALS AND METHODS

train and Plasmids

E. coli JM109(DE3) has the following genotype: endA1,ecA1, gyrA96, thi, hsdR17(rk2,mk1), relA1, supE44,(lac-proAB), (F9, traD36, proAB, lacIqZDM15), l(DE3).2 Abbreviations used: LeuRS, leucyl-tRNA synthetase; IPTG, iso-

ropyl-b-D-thiogalactopyranoside; CD, circular dichroism.

115

116 CHEN ET AL.

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117PURIFICATION OF LEUCYL-tRNA SYNTHETASE

he plasmid pMFT7-5 was a T7 promoter-based expres-ion vector, which was featured by a strong T7 promoter,erminator, translations start, a multiple cloning site, anmpicillin resistance marker, and an f1 phage origin (16).he plasmid pTrc-leuS2 contains the gene encoding E.oli LeuRS (17).

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Oligonucleotides for constructing the plasmid wereynthesized at Shanghai Institute of Plant Physiology,cademia Sinica. Restriction endonucleases and T4 DNA

igase were the products of Boehringer MannheimmbH. Isopropyl-b-D-thiogalactopyranoside (IPTG)nd imidazole were purchased from Sango. Ni-NTAuperflow was obtained from QIAGEN.

NA Recombinant Techniques

The DNA manipulations were conducted as de-cribed (18).

xpression of the Cloned Gene

An overnight starter culture of E. coli strain JM109(DE3)ransformants containing the recombinant plasmidas diluted to a final concentration of 5% in 0.5 liter ofuria–Bertani medium containing 100 mg/ml ampicil-

in and allowed to grow at 37°C with vigorous shaking300 rpm). When an OD600 of 0.5 with the culture waseached, IPTG was added to a final concentration of 0.5M, and the incubation continued under the same

ondition for another 4–5 h. Then, the cells were har-ested by centrifugation at 4000g for 10 min (4°C). Theell pellet can be stored at 270°C if not used instantly.

urification of His6-LeuRS

Two grams of wet cells from 500 ml culture wereuspended in 8 ml lysis buffer (50 mM NaH2PO4, pH

TAB

Purification of the His6-LeuRS from

StepProtein

(mg)Total activity

(units)

rude extract 120 39840i-NTA 14.2 21854

a Enzyme was purified from about 2 g of wet cells.

IG. 1. Schematic representation of the construction of the plasontained NdeI (underlined with single solid line at the 59 end), EcoRestriction site (underlined with dashed line). The initiation codon And then the synthetic oligo was inserted between the two sites. Thiontaining leuS gene from the plasmid pTrc-leuS2 was isolated by 0.etween the NcoI and HindIII sites. This yielded plasmid pMFT7H6-

nderlines respectively. Initiation codon (ATG) and end codon (TAA) ar

.0; 300 mM NaCl; 10 mM imidazole) and sonicated once. The lysate was centrifuged at 27,000g for 30 min at°C to pellet the cellular debris. The recovered super-atant was applied onto a Ni-NTA Superflow column1 3 3 cm) equilibrated with lysis buffer and washedith the same buffer until OD280 is constant. Afternother washing with 20 ml wash buffer (50 mMaH2PO4, pH 8.0; 300 mM NaCl; 20 mM imidazole),

he enzyme was eluted with an elution buffer (50 mMaH2PO4, pH 8.0; 300 mM NaCl; 100 mM imidazole).he fraction containing the synthetase was pooled andoncentrated by dialysis against 10 mM potassiumhosphate buffer, pH 6.8, containing 50% glycerol. Theurified enzyme could be stored in the above buffer at20°C and remained stable for at least 6 months.

nzyme Assay

The aminoacylation activity of LeuRS was deter-ined as described (19). One unit of aminoacylation

ctivity was defined as the amount of enzyme whichharged 1 nmol of tRNALeu per minute under a givenondition. The kinetic constants of LeuRS were deter-ined using various concentrations of the relevant

ubstrates. Protein concentration was measured by theethod of Bradford (20). Polyacrylamide gel electro-

horesis with 10% separating gel and 4% stacking geln the present of sodium dodecyl sulfate (SDS–PAGE)as performed as described (18).

ircular Dichroism (CD) Spectroscopy

Protein samples with concentration of 0.2 mg/mlere measured on a Jasco J-715 spectropolarimeterith nitrogen purge at room temperature. A 0.1-cmathlength cuvette was used and spectra were accu-ulated over five scans.

1

coli JM109(DE3)/pMFT7H6-LeuRSa

Specific activity(units/mg)

Recovery(% total) Purification fold

332 100 11539 55 4.6

pMFT7H6 and pMFT7H6-LeuRS. The synthetic oligonucleotidesnderlined by double solid line at the 39 end) cohesive ends, and NcoIis boxed. The plasmid pMFT7-5 was digested with NdeI and EcoRI

ielded pMFT7H6. The 2.6-kb NcoI–HindIII-digested DNA fragmentagarose gel electrophoresis and inserted into the plasmid pMFT7H6

RS. The NcoI and HindIII sites were marked by single and dashed

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ESULTS

onstruction of the Plasmids pMFT7H6 andpMFT7H6-LeuRS

The construction of His6-tagging vector pMFT7H6 ishown schematically in Fig. 1. In brief, synthetic oli-onucleotides with sequences encoding six histidineesidues were inserted into the polylinker region of thelasmid pMFT7-5 between NdeI and EcoRI restrictionites to yield the His6-tagging vector. The sequence ofhe insertion was confirmed by DNA sequencing (dataot shown). Besides the His6 domain, the inserted oli-os also provided a start codon (ATG) and NcoI site forhe cloning of the target gene encoding LeuRS. Toinimize the unfavorable effects of the His6-tag on the

usion protein, the inserted oligos also encoded a flex-ble peptide, GlyGlyAla, between His6 domain and tar-et protein. The resulting plasmid pMFT7H6 can directhe synthesis of a fusion protein with an oligo–Hisomain at the N terminus and overproduce the targetrotein with its highly efficient expression elements.The gene encoding LeuRS was cut out from the plas-id pTrc-leuS2 by NcoI and HindIII and then inserted

nto the plasmid pMFT7H6 between NcoI and HindIIIestriction sites to generate the plasmid pMFT7H6-euRS (Fig. 1), from which the LeuRS fused with andditional tag, Met His His His His His His Gly Glyla, at the N terminus (His6-LeuRS) was overpro-uced.

xpression and Purification of His6-LeuRS

The His6-LeuRS overproduced in the E. coli strainM109(DE3) transformant containing pMFT7H6-LeuRS

IG. 2. SDS–PAGE analysis of the synthetase in the course of theurification. Electrophoresis was on a 15% running gel and proteinsere visualized by Coomassie staining. Lane 1, protein standardsith molecular weights of 205.0, 116.0, 97.4, and 66.0 kDa, respec-

ively. Lane 2, induced cells containing about 5 mg protein sample.ane 3, 5 mg purified synthetase after Ni-NTA Superflow.

as subjected to one-step chromatography on Ni-NTA w

uperflow and the fusion protein was fractionated to aurity over 90% (Table 1). The specific activity of theynthetase in the crude extract of the transformantM109(DE3)/pMFT7H6-LeuRS was approximately 110imes that of JM109(DE3) (the host strain without theector), and about 14.2 mg purified enzyme was ob-ained from 2 g of wet cells. The purified enzyme hashe same molecular weight (about 98.5 kDa) as calcu-ated according to its amino acid sequence and as de-ermined by SDS–PAGE (Fig. 2). The specific activityf His6-LeuRS was 1539 units/mg, in the same range ashe 1690 units/mg for LeuRS.

inetics of His6-LeuRS and LeuRS

The kinetic constants of the aminoacylation reactionatalyzed by His6-LeuRS and LeuRS for the three sub-trates are shown in Table 2. There are no significant

IG. 3. CD spectra of His6-LeuRS (dashed line) and LeuRS (solidine). Protein sample concentration was 0.2 mg/ml. A 0.1-cm path-ength cuvette was used and spectra were accumulated over fivecans. The wavelength range varied from 250 to 190 nm. Ellipticity

TABLE 2

Kinetic Constants of His6-LeuRS and LeuRSa

Substrates Constants His6-LeuRS LeuRS

Km (mM) 15 15eucine kcat (s21) 3.0 3.4

kcat /Km (s21mM21) 202 227Km (mM) 280 260

TP kcat (s21) 3.6 4.2kcat /Km (s21mM21) 13 16Km (mM) 1.6 1.6

RNALeu kcat (s21) 3.4 3.9kcat /Km (s21mM21) 2125 2438

a All data in this table were the average values from three inde-endent determinations.

as expressed as mean residue weight ellipticity.

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119PURIFICATION OF LEUCYL-tRNA SYNTHETASE

hanges in the Km and kcat values of His6-LeuRS foreucine, ATP, and tRNALeu compared with those ofeuRS. The result showed that the additional shorteptide at the N terminus of His6-LeuRS had littleffect on the binding of its substrates, the rate of ami-oacylation, and its catalytic efficiency.

D Spectra of His6-LeuRS and LeuRS

The CD spectra of His6-LeuRS and LeuRS are pre-ented in Fig. 3. The two curves display little differenceetween them, indicating that the oligo–His domain athe N terminus of LeuRS did not affect its secondarytructure.

ISCUSSION

To obtain a sufficient amount of material for studiesn the structure and function relationship of an en-yme, molecular cloning of its gene is the most usedrocedure. The best system should produce the ex-ressed protein not only in high yield but also easilyurifiable. One system that fulfills the above criteria ismmobilized metal affinity chromatography, which has

any advantages of high-binding capacity, mild elu-ion condition, and stability. Compared with other af-nity chromatography, the His6-fusion protein canind to the Ni-NTA groups on the matrix (Ni-NTAuperflow) with an affinity greater than that of anti-ody–antigen or enzyme–substrate interaction (21,22).esides, the six histidine residues at the N terminus of

he fusion protein are much smaller than those largeolypeptides fused at the N terminus of the fusionrotein in other affinity methods (23,24), the unfavor-ble effect on the structure and activity of target en-yme will be minimized, and the His6-tag does not needo be removed from the purified fusion protein.

So far some His6-fusion proteins have been crystal-ized successfully (25). The overexpressed His6-LeuRSould be purified to a purity above 90% after one-stephromatography on Ni-NTA Superflow, which wasure enough for crystallization. Furthermore, becausehe purification method was fast, the recombinant pro-ein could be kept stable during purification, especiallyor some unstable mutants. It provided great conve-ience for the crystallization to solve its X-ray struc-ure.

CKNOWLEDGMENTS

This work was supported by the Natural Science Foundation ofhina (Grant 39730120) and the Chinese Academy of Sciences

Grant KJ951-B1-610). We are grateful to Prof. Bo-Liang Li for theMFT7-5 plasmid.

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