3rd

Cloning and Expression in Escherichia coli of theRecombinant His-Tagged DNA Polymerases fromPyrococcus furiosus and Pyrococcus woesei

Sławomir Dabrowski and Jozef Kur1

Department of Microbiology, Technical University of Gdansk, ul. Narutowicza 11/12, 80-952 Gdansk, Poland

Received May 6, 1998, and in revised form June 25, 1998

Complete PCR-derived DNA fragments containingthe structural genes for DNA polymerases of the ar-chaeons Pyrococcus furiosus and Pyrococcus woeseiwere cloned into an expression vector. The clones ex-pressing thermostable His-tagged DNA polymeraseswere selected. The cloned fragments were sequenced.The DNA sequences were verified to be authentic bysequencing several clones. The nucleotide (nt) se-quence revealed that DNA polymerase of P. woesei(Pwo DNA polymerase) consists of 775 amino acids andhas a molecular weight of 90,566. It shows 100% nucle-otide identity to the nucleotide sequence of DNA poly-merase from P. furiosus (Pfu DNA polymerase). Theresults confirm that nucleotide sequences of both ar-chaeons (P. furiosus and P. woesei) are highly similar.The recombinant DNA polymerases (His-tagged Pfuand His-tagged Pwo) contained a polyhistidine tag atthe N-terminus (43 additional amino acids) that al-lowed single-step isolation by Ni-affinity chromatogra-phy. We found that recombinant plasmids are toxic orunstable in the expressing strain BL21(DE3), even inthe absence of the inducing agent, IPTG. However, theplasmids were stable in BL21(DE3) containing thepLysS plasmid, which suppresses expression prior toinduction, and His-tagged proteins were expressedupon IPTG addition. The proteins were purified byheat treatment (to denature E. coli proteins), followedby metal-affinity chromatography on Ni21-Sepharosecolumns. The enzymes were characterized and dis-played high DNA polymerase activity and thermosta-bility. This bacterial expression system appears to bethe method of choice for production of Pfu or PwoDNA polymerases. © 1998 Academic Press

Key Words: PCR; DNA polymerase; recombinant pro-tein; metal-affinity chromatography; DNA sequence.

A number of thermophilic DNA polymerases havebeen isolated previously and characterized from bothmesophilic eubacteria and archaea sources. Those thathave been analyzed are monomeric in solution withmolecular masses of 80–115 kDa (1–3). As expected,these enzymes have elevated temperature optima andhave thermal stabilities that roughly correspond to thethermal extremes of the environment from which theywere isolated. Despite the fact that thermal stabilitiesof the native proteins vary among the enzymes, anoptimal temperature for polymerization of 70–80°C iscommon. As has been pointed out by many authors,this suggests that the template stability rather thanthe intrinsic enzyme stability determines the optimaltemperature for polymerization (2,3). Due to the ther-mostability of those enzymes, the structure and func-tion relationships and the potential industrial applica-tions of many thermostable enzymes such as DNApolymerases are of considerable interest to research-ers. However, most native thermostable enzymes aresynthesized at very low levels by the thermophilic bac-teria and are therefore cumbersome to purify.

More than 50 DNA polymerase genes have beencloned and sequenced from various organisms, includ-ing thermophiles and archaea. Amino acid sequencesdeduced from their nucleotide sequences can be classi-fied into four major groups: Escherichia coli DNA poly-merase I (family A), DNA polymerase II (family B,a-like DNA polymerases), DNA polymerase III (familyC), and others (family X) (4). In 1975 a new concept foraffinity purification of proteins was presented byPorath and co-workers (5). The method is based on theinteraction between the side chains of certain aminoacids, particularly histidines, on a protein surface andthe immobilized transition of metal ions, and is knownas immobilized metal ion affinity chromatography(IMAC). The metal ions are immobilized by the use of achelating agent capable of presenting the metals for

1 To whom correspondence should be addressed. E-mail: [email protected].

PROTEIN EXPRESSION AND PURIFICATION 14, 131–138 (1998)ARTICLE NO. PT980945

1311046-5928/98 $25.00Copyright © 1998 by Academic PressAll rights of reproduction in any form reserved.

binding to the protein. Several gene fusion systemsemploying histidine-rich tags for purification of recom-binant proteins have been described so far. The tags,either N- or C-terminal, consisting of consecutive his-tidine residues binding selectively to immobilized Ni21

ions were described by Houchuli and co-workers (6).The adsorption of the poly-His-tagged proteins to ametal-chelate adsorbent was performed at neutral orslightly alkaline pH, at which the imidazole group ofhistidine is not protonated (7). The expressed fusionproteins were recovered with purity of more than 90%in a single step using a Ni21-nitrilotriacetic acid (NTA)adsorbent and elution with low pH or by competitionusing imidazole (6,7). The use of polyhistidine domainsas fusion tags for protein purification has been demon-strated for recombinant fusion proteins produced in awide range of host cells including E. coli, Saccharomy-ces cerevisiae, mammalian cells, and baculovirus-infected Spodoptera frugiperda insect cells (8–13). Theuse of polyhistidines as fusion tags has been demon-strated also for the purification of active Taq and Tththermostable DNA polymerases (14; Dabrowski andKur, manuscripts in preparation). An important ad-vantage of His6 affinity tag is the possibility of purify-ing proteins under denaturing conditions, thus facili-tating purification of less soluble proteins.

Extremely thermostable DNA polymerases havebeen purified from some archaeons (1–3,15) and thegenes have been cloned (16,17). The deduced aminoacid sequences of the DNA polymerases showed thatthey all belong to the a family. The DNA polymerasefrom Pyrococcus furiosus (Pfu) has gained considerableattention in the field of DNA amplification. It is athermophilic DNA polymerase with 39-59 exonucleaseactivity that corrects errors introduced during the po-lymerization. Several thermophilic DNA polymerases,including Pfu, Vent, Pwo, and UlTma, also have proof-reading ability, but they differ in their error rate (18,–21). The error rate for Pfu is reported to be 7- to 10-foldlower than that of nonproofreading Taq DNA polymer-ase, and 2- to 30-fold lower than other proofreadingenzymes (21–23). The Pfu DNA polymerase was ini-tially characterized for the preparation isolated di-rectly from P. furiosus (16), but this thermophilic, an-aerobic bacterium is difficult to grow so as to obtainlarge amounts of protein. A major advance was expres-sion of recombinant Pfu DNA polymerase in a baculo-virus-mediated system (24). This system makes possi-ble production of commercial amounts, but is not ascheap and convenient as an E. coli bacterial expressionsystem. The Pfu DNA polymerase was also produced inE. coli expression system as a native protein givingmilligram quantities of enzyme (25).

The results of this study demonstrate that His6-tagged Pfu and Pwo DNA polymerases can be effi-ciently synthesized in a biologically active form in the

E. coli overexpression system and that large amountsof active enzyme can be purified using a Ni21-Sepha-rose single-step chromatography procedure. We reportalso the previously unknown nucleotide sequence ofthe P. woesei DNA polymerase gene and the deducedamino acid sequence of its protein.

MATERIALS AND METHODS

Bacterial Strains, Plasmids, Enzymes, and Reagent

Pyrococcus furiosus (DSM 3638) and P. woesei (DSM3773) strains were obtained from DSMZ-DeutscheSammlung von Mikroorganismen und ZellkulturenGmbH (Braunschweig, Germany) and were used assources of the total DNA for PCR amplifications. The E.coli DH5a strain was used for preparation of plasmidsand cloning, and BL21 (DE3) pLysS (Promega, U.S.A.)was applied to expression of His6-tagged Pfu and PwoDNA polymerases. The plasmid pET30-LIC (Novagen,UK) was used for the construction of the expressionsystem. The E. coli cells with plasmids were culturedaerobically at 37°C in LB medium supplemented witha 34 mg/ml kanamycin for the DH5a strain or with 50mg/ml chloramphenicol and 34 mg/ml kanamycin forthe BL21 (DE3) pLysS strain. Restriction and modifi-cation enzymes were purchased from Promega. Thereagents for PCR and Ni21-TED Sepharose columnswere obtained from DNA-Gdansk (Poland). IPTG, aga-rose, and reagents for protein purification were pur-chased from Sigma.

Cloning of the DNA Polymerase Genesfrom P. furiosus and P. woesei

The P. furiosus or P. woesei cells were grown at 95°Cwithout shaking, in broth as described for P. furiosusby Uemori et al. (26). DNA was isolated using aGenomic DNA Prep Kit (A&A Biotechnology, Poland).The high homology between the known DNA andamino acid sequences of P. furiosus and P. woesei (Ta-ble 1) suggests that both archaeon are very similar.Based on the known DNA sequence of Pfu DNA poly-merase gene (GenBank Accession No. D12983) the spe-cific primers for Pfu pol gene were synthesized andused for the amplification from DNA templates of P.furiosus and P. woesei. The PCR conditions were firstdesigned for the efficient amplification of the Pfu DNApolymerase gene and then applied to the amplificationof Pwo DNA polymerase gene. The reaction solutionconsisted of P. furiosus or P. woesei DNA (100 ng), 2 ml(10 mM) of each primer (Del1-LIC 59 GAC GAC GACAAG ATG ATT TTA GAT GTG GAT TAC 39; Del2-LIC59 GAG GAG AAG CCC GTT GAT ATC TAT CGC TTTTCT AGG A 39), 5 ml (10 mM) dNTP’s, 5 ml 10 3 PCRbuffer (100 mM Tris-HCl, pH 8.9, 500 mM KCl, 20 mMMgCl2, 1% Triton X-100), and 2 u of thermostable Pfu

132 DABROWSKI AND KUR

DNA polymerase (Stratagene); 30 cycles were per-formed with a temperature profile of 1 min at 93°C, 1min at 45°C, and 2 min at 72°C in a Hot-Shot12 ther-mal cycler (DNA Gdansk, Poland). The amplificationproducts were analyzed by electrophoresis on a 1%agarose gel stained with ethidium bromide.

Specific, approximately 2370-bp PCR products wereobtained for Pfu DNA polymerase as well as Pwo DNApolymerase gene. Both products gave the same electro-phoretic pattern after restriction endonucleases EcoRI,HinfI, HindIII, BamHI, and MspI digestions (data notshown).

The PCR products were cloned using the ligase-independent cloning (LIC) system (Novagen, UK) intopET30-LIC plasmid expression vector (Novagen, UK).First, a PCR product from P. furiosus or P. woeseitemplates was purified from an agarose gel slice afterelectrophoresis using Gel Out kit (A&A Biotechnology)and treated with T4 DNA polymerase in the presenceof dATP for ligation-independent cloning into thepET30-LIC plasmid expression vector. The plasmidswere initially propagated in the E. coli DH5a and theselected clones having recombinant plasmids withlarge inserts were digested with EcoRI, BamHI, andHindIII, so as to confirm the identity of the cloned DNAfragments.

Purification of Recombinant His6-Tagged Pfu or PwoDNA Polymerase

Escherichia coli strain BL21(DE3) pLysS trans-formed with pET30pfu or pET30pwo was grown at37°C in 1000 ml LB containing 50 mg/ml chloramphen-icol and 34 mg/ml kanamycin to an A660 of 0.3. IPTGwas then added to the final concentration of 1 mM. Thecells were harvested after 4 h by centrifugation (9 g ofcell mass) and the pellet was resuspended in 20 ml ofbuffer B (20 mM Tris, pH 7.9, 500 mM NaCl, 5 mMimidazole, 0.1% Triton X-100). The cells were disruptedby sonication, and the insoluble debris was removed bycentrifugation. For purification the cleared lysate wasimmersed in a 75°C water orbital shaker and shaken

for 30 min, cooled on ice for 20 min, and then cen-trifuged at 16,000g at 4°C for 20 min. The clarifiedsupernatant (about 19 ml) was then applied directlyonto a Ni21-TED (tris(carboxymethyl)ethylenediamine)Sepharose column (10 ml of bed volume; DNA-Gdansk)preequilibrated with 4 vol of buffer B. After loading,the column was washed several times with 10 ml of thesame buffer until the UV absorption returned to thebaseline. His6-tagged Pfu/Pwo DNA polymerase waseluted three times with 10 ml of washing buffer Bcontaining 40, 60, and 100 mM imidazole, respectively(buffers B40, B60, and B100). The eluted fractionswere pooled and dialyzed against a buffer containing20 mM Tris, pH 7.9, 100 mM KCl, 0.1% Triton X-100,0.1 mM EDTA, 1 mM DTT, and 50% glycerol. Theconcentration of purified enzymes was determinedfrom ultraviolet absorption, using the extinction coef-ficient for A280 5 0.78 for 1 mg/ml, calculated from thenumber of trp and tyr residues in the sequence (usingthe Protean program of DNAstar, Madison, WI) andalso estimated by the Pierce BCA assay using BSA asa standard.

The native Pfu or Pwo DNA polymerase enzyme wasobtained by proteolytic cleavage of 3 mg of recombinantHis6-tagged DNA polymerase with 0.05 u of enteroki-nase (Novagen, UK) in a buffer containing 20 mMTris-HCl, pH 7.4, 50 mM NaCl, and 2mM CaCl2 at23°C for 16 h.

Determination of Pfu and Pwo DNA PolymerasesGenes’ Nucleotide Sequences

The independently derived clones expressing thermo-stable DNA polymerase were selected and sequenced us-ing an ALFexpress DNA sequencer (Pharmacia Biotech,Sweden) using an ALFexpress AutoRead sequencing kit(Pharmacia Biotech, Sweden). Both strands were readentirely and 2370 bases were determined.

Assays for Relative DNA Polymerase Activity

The relative DNA polymerase activities were deter-mined by comparing band intensities of the PCR-

TABLE 1

Identity of Known Nucleotide and Protein Sequences of the Archaebacteria Pyrococcus furiosus and Pyrococcus woesei

Aligned gene sequencesGenBank Accession No.

[P. furiosus (DSM3638)/P. woesei (DSM 3773)]

Identity ofnucleotide sequence

(%)

Identity ofprotein sequence

(%)

Transcription factor TFIIB U48391 /X70668 100 100TATA-box binding protein U48392 /U10285 100 100Glutamine synthetase glnA L12410 /X60161 98.6 97DNA polymerase D12983 /U84155a 100 100b-Galactosidase E08095 /AF043283 99.9 99.8

a This study.

133CLONING AND EXPRESSION OF DNA POLYMERASES

amplified DNA obtained with His6-tagged Pfu, His6-tagged Pwo, the native Pfu (Stratagene), and thenative Pwo (Boehringer Mannheim) DNA polymerases.The PCR system of bovine leukemia virus (BLV) detec-tion and PCR conditions used in this assay were asdescribed by Kubis et al. (27) with some modifications.The primers (ZM4-59 CTC GCC CTC CCG GAC GCCCA and ZM5 - 59 GCT AGG CCT AAG GTC AGG GCCGC) were applied to amplify a 218-bp fragment of theenv virus gene. The pENV1 plasmid was used as thematrix DNA. A 1-ml portion of plasmid DNA (10 pg)was combined with 2.5 ml of 103 reaction buffer (500mM KCl, 100 mM Tris-HCl, pH 8.8, 25 mM MgCl2, 1%Triton X-100), 2.5 ml of a deoxynucleosides triphos-phate (dNTP) mixture (2.5 mM of each dNTP), 1 ml ofeach primer (10 mM), and 16 ml of water. This mixturewas supplemented with different volumes of recombi-nant enzyme fraction (final preparate of the His6-tagged Pfu or Pwo polymerase) or 1 u of the native Pfuor Pwo DNA polymerase. The amplification reactionswere performed with an automated thermocycler (ther-mocycler Hot-Shot 25, DNA-Gdansk) according to thefollowing scheme: an initial cycle of 1 min at 94°C wasfollowed by 30 cycles of denaturation for 30 s at 94°C,annealing and elongation of primers for 1 min at 64°C,and an extension step at 72°C for 5 min.

PCR-specific products were separated electrophoreti-cally on a 2% agarose gel using 13 Tris-borate EDTA

running buffer at a field strength of 8 V/cm. The DNAwas visualized on an ultraviolet trnsilluminator follow-ing ethidium bromide staining and photographed.

The Examination of His6-Tagged Pfu or PwoPolymerase Thermostability

For the thermostability studies, His6-tagged Pfu orPwo polymerase was heat-treated at 98°C for 1, 2, 4, 6,and 8 h before PCR reaction. After various incubationperiods the enzyme samples (1 unit) were withdrawnand tested for the DNA polymerase activity using spe-cific PCR amplification with pENV1 matrix as de-scribed above.

RESULTS AND DISCUSSION

Construction of the Recombinant Vectors ProducingHis6-Tagged Pfu or Pwo DNA Polymerase

The pET30-LIC system is one of the most powerfulsystems developed for cloning and expression of recom-binant proteins in E. coli (28). The pET30-LIC vectorhas a very strong T7 promoter and can be grown incombination with pLysS to provide additional strin-gency (29,30). We amplified the pfu or pwo gene usingthe proofreading Pfu DNA polymerase (Stratagene).The amplification product correspondng to the full-sizegene (2370 bp) of P. furiosus or P. woesei DNA poly-

FIG. 1. The sequences of primers, nucleotides, and amino acids of the pETpfu and pETpwo plasmids near the site of the polymerase geneinsertion.


merase was treated with T4 DNA polymerase in thepresence of dATP so as to create LIC ends, and insertedinto pET30-LIC vector, thus obtaining the recombi-nant pET30pfu or pET30pwo plasmid. The nucleotidesequences of these constructs were confirmed by se-quencing analysis to ensure that the reading frame iscorrect. The primer, nucleotide, and amino acid se-quences of the pETpfu or pETpwo plasmid near the siteof the DNA polymerase gene insertion are shown inFig. 1. The obtained genetic constructs retained theopen reading frame, and the target proteins contained43 additional amino acid residues at N-terminus, in-cluding a cluster of six histidine residues for purifica-tion of the recombinant proteins by metal-affinity chro-matography.

Determination of DNA Pwo pol Gene Sequence

All clones of pET30pwo exhibited the same nucleo-tide sequence of pol gene as was determined by se-quencing using an ALFexpress DNA sequencer and anALFexpress AutoRead sequencing kit (Pharmacia Bio-tech). The alignment analyses of the obtained DNAPwo pol gene sequence and known DNA Pfu pol genesequence performed with NALIGN program (PCGENEpackage) revealed 100% identity. The results of oursequencing data confirm that P. furiosus and P. woeseiare closely related organisms and were comparable tothe identities obtained in the alignments within thegroup of known sequences for P. furiosus and P. woesei(see Table 1).

The complete sequence of P. woesei DNA polymerasegene can be obtained from GenBank (Accession No.U84155).

Expression of the Recombinant His6-Tagged Pfuor Pwo DNA Polymerases

The level of expression was examined in differentstrains. When pETpfu or pETpwo was transformed intoE. coli BL21(DE3) at 37°C no colonies were obtained.This observation suggested that pETpfu or pETpwo plas-mide might produce small amounts of recombinant poly-merase prior to IPTG induction with toxic effect to E. coli.The recombinant plasmid pET30pfu or pET30pwo wasthen transformed into E. coli BL21(DE3) pLysS. ThepLysS plasmid, which expresses T7 lysozyme in the bac-terial cytoplasm, strongly represses protein expressionfrom the pET vectors in the absence of induction, thus

FIG. 2. SDS electrophoresis in 10% polyacrylamide gel of the fractions obtained by purification of the recombinant His6-tagged Pfu DNApolymerase (A) and His6-tagged Pwo DNA polymerase (B) from E. coli BL21 (DE3) pLysS. Lanes 1, midrange molecular weight marker(Promega); lanes 2, lysate from E. coli BL21(DE3) pLysS; lanes 3, lysate from E. coli BL21 (DE3) pLysS 1 pETpfu (A) or pETpwo (B); lanes4, cleared lysate after heat treatment (53 concentrate); lanes 5 and 6, flowthrough fraction with the B buffer; lanes 7, washed fraction withthe B20 buffer; lanes 8, eluted fraction with the B40 buffer; lanes 9, eluted fraction with the B60 buffer; lanes 10, eluted fraction with theB100 buffer.

FIG. 3. Monitoring of the recombinant His6-tagged Pfu DNA poly-merase activity (A) and His6-tagged Pwo DNA polymerase activity(B) of the fractions obtained during purification procedures usingspecific PCR reaction. Lanes 1 represent the molecular weightmarker (501, 489, 404, 331, 242, 190, 147, 111, 110 bp); lanes 2, 0.1ml of the clarified supernatant after heat treatment; lanes 3 and 4, 1ml of the flowthrough fraction with the B buffer; lanes 5, 1 ml of thefraction washed with the B20 buffer (20 mM imidazole); lanes 6, 1 mlof the fraction eluted with the B40 buffer (40 mM imidazole); lanes 7,1 ml of the fraction eluted with the B60 buffer (60 mM imidazole);lanes 8, 1 ml of the fraction eluted with the B100 buffer (100 mMimidazole).


enabling expression of very toxic proteins. The mecha-nism of the additional stringency is that the T7 lysozymebinds to and inactivates T7 RNA polymerase (which isexpressed at low levels in the absence of induction) andinhibits transcription. Following induction, the amountof T7 RNA polymerase is sufficient to overcome the ly-sozyme inhibition. We found that colonies could easily beobtained at 37°C when pETpfu or pETpwo was used totransform the E. coli BL21(DE3) carrying pLysS plas-mid. Bacterial colonies grown on chloramphenicol andkanamycin-containing agar were assayed for DNA poly-merase activity using specific PCR amplification with thepENV1 matrix. The clones displaying high DNA poly-merase activity were selected. The selected clone wasgrown in LB medium supplemented with the chloram-phenicol and kanamycin. Overexpression was induced byIPTG addition (final concentration, 1 mM) at variousstages of culture (A660: 0.1, 0.2, 0.3, 0.4, 0.5, and 0.6). Thebest induction of His6-tagged Pfu or Pwo DNA polymer-ase synthesis was obtained at A660 of 0.3. At A660 . 0.4the synthesis of the recombinant protein and DNA poly-merase activity were significantly lower.

Purification of the Recombinant His6-Tagged Pfuor Pwo DNA Polymerases

The purity of enzymes was examined by sodium do-decyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE) (Fig. 2). Monitoring of the His6-tagged Pfu orPwo DNA polymerase activity was carried out by PCR-specific reaction with 1 ml of protein fractions with-drawn during purification procedure (Fig. 3). His6-tagged Pfu or Pwo DNA polymerases were expressed asa soluble form in the cytosol. Before the chromatogra-phy step, the cell lysate was heat treated at 75°C for 30min. The heat denaturation step resulted in the pre-cipitation of the vast majority of contaminating cellularproteins (Figs. 2A and 2B, lanes 4). Several E. coliproteins still remained soluble after the heating step.

However, at this stage, His6-tagged Pfu or Pwo DNApolymerases were pure enough for PCR applications.For the final purification, the clarified medium waschromatographed on a Ni21-TED Sepharose column.The recombinant enzymes were eluted from the col-umn as a single peak (about 97% purity) (Figs. 2A and2B, lanes 6–8). A band corresponding to a 95-kDaprotein was observed in SDS–PAGE of crude extractsof E. coli BL21(DE3) pLysS 1 pET30pfu or E. coliBL21 (DE3) pLysS 1 pET30pwo cultures after IPTGinduction (Figs. 2A and 2B, lanes 3). This band wasabsent in the control crude extracts of E. coli BL21(DE3) pLysS cultures (Figs. 2A and 2B, lanes 2). Thepronounced enrichment of the 95-kDa protein depictedin Fig. 2 (lanes 4) after the heat denaturation step(compare lane 4 with lane 3 in Figs. 2A and 2B) shouldbe noted. The amount of the 95-kDa protein, and con-sequently the specific activity, was estimated by ultra-violet absorption (A280) determination or using BSA asa standard (see Table 2). We obtained 24 mg of purifiedHis6-tagged Pfu polymerase or 26.6 mg of purifiedHis6-tagged Pwo, as determined by the A280. After the

FIG. 4. The DNA polymerase activity of the His6-tagged Pwo DNApolymerase after purification by affinity chromatography on theNi21-TED Sepharose (DNA-Gdansk) determined using specific PCRamplification. Lane 1, the molecular weight marker (501, 489, 404,331, 242, 190, 147, 111, 110 bp); lanes 2–7, purified enzyme of 2, 1,0.5, 0.3, 0.2, and 0.1 ml, respectively; lane 8, 1 u of the commercialPwo DNA polymerase (Boerhringer Mannheim).

TABLE 2

Purification of His6-Tagged Pfu or Pwo DNA Polymerase Expressed in E. coli

Purification step

Total protein a (mg) Specific activity (units/mg) Purity (%)b

His6-taggedPfu

His6-taggedPwo

His6-taggedPfu

His6-taggedPwo

His6-taggedPfu

His6-taggedPwo

Total bacterial lysatec ND ND ND ND 6 7Heat-soluble proteins 60.0 63.3 ND ND 40 42Metal-affinity chromatography 24.0 26.6 31,000 31,000 97 97

a The concentration of heat-soluble proteins was determined by the Pierce BCA assay using BSA as a standard. The concentration ofprotein after metal affinity chromatography was determined from ultraviolet absorption, using the extinction coefficient for A280 5 0.78 for1 mg/ml, calculated from the number of trp and tyr residues in the sequence (using the Protean program of DNAstar, Madison, WI).

b Purity of enzymes at each step was estimated by analyzing images of the SDS–PAGE gel stained with Coomassie blue using BiometraScanPack software. ND, not determined.

c Nine grams of the wet weight of cells used in the preparation.


dialysis of recombinant proteins against storage buffer,the DNA polymerase activity was 2.8 units/ml (i.e.,746,000 units/liter medium, 31,000 units/mg) for thepurified His6-tagged Pfu DNA polymerase and 3.1units/ml (i.e., 827,000 units/liter medium, 31,000 units/mg) for the purified His6-tagged Pwo DNA polymeraseisolated from 1-liter E. coli cultures (see also Table 2).We have found that the reproducible high yields ofHis6-tagged enzymes are obtained from freshly trans-formed cells, whereas the expression from a freezerstock gave erratic and lower yields.

Examination of the Relative DNA Polymerase Activity

The relative DNA polymerase activity was deter-mined by comparing the PCR results obtained for theHis6-tagged Pfu or Pwo DNA polymerase and the na-tive Pfu DNA polymerase (Stratagene) (results notshown) or the native Pwo DNA polymerase (Boehr-inger Mannheim) (Fig. 4) using specific PCR amplifi-cation of the BLV env gene target. As shown in Fig. 4,the relative His6-tagged Pwo DNA polymerase activitywas about 3.1 u/ml and about 2.8 u/ml for His6-taggedPfu DNA polymerase (results not shown).

The relative His6-tagged Pfu or Pwo DNA polymer-ase activity was also comparable to the His6-tagged Pfuor Pwo DNA polymerase cleaved with enterokinase(result not shown) using the same specific PCR ampli-fication test.

Thermostability

His6-tagged Pfu or Pwo DNA polymerase was verystable and active at high temperature. The thermalinactivation level at 98°C of the purified His6-taggedPwo enzyme is shown in Fig. 5.

CONCLUSIONS

1. His6-tagged Pfu or Pwo DNA polymerase expressedin E. coli can easily be prepared in milligram quantities,and it appears to have the same activity as that purified

from native sources or that expressed in the baculovirussystem in case of Pfu DNA polymerase.

2. His6-tagged Pfu or Pwo DNA polymerase was pu-rified in a single chromatography step using a Ni21-TED Sepharose column (97% purity).

3. The 43 additional N-terminal amino acid resi-dues (including a cluster of six histidine residues forpurification of the recombinant protein by metal-affinity chromatography) have no effect on polymer-ase activity.

4. The effective cloning of the PCR products ob-tained using His6-tagged Pfu or Pwo DNA polymeraseinto blunt-ended vectors suggested the presence of39-59 exonuclease activity in the recombinant proteins.

5. The applied overexpression system was very effi-cient, giving 31,000 units/mg of the purified His6-tagged Pfu or His6-tagged Pwo DNA polymerase activ-ity. This is similar to the activity reported for Pfudirectly purified from P. furiosus (31,713 units/mg(15)), from baculovirus expression (26,000 units/mg(24)), or from the bacterial pET11 expression system(22,500 units/mg (25)).

6. The purified recombinant enzymes exhibited highpolymerase activity and high thermostability.

ACKNOWLEDGMENTS

The work was supported by the Technical University of Gdanskand DNA-Gdansk II s.c.

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FIG. 5. Temperature stability assay. The His6-tagged Pwo DNApolymerase (1 u, 0.032 mg) was heat treated at 98°C before PCRreaction for 0 h (lane 2), 1 h (lane 3), 2 h (lane 4), 4 h (lane 5), 6 h (lane6), and 8 h (lane 7). Lane 1, the molecular weight marker (501, 489,404, 331, 242, 190, 147, 111, 110 bp). PCR products were run on a 2%agarose gel, stained, and photographed under UV illumination.


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