PROTEIN EXPRESSION AND PURIFICATION ARTICLE NO.

14, 131138 (1998)

PT980945

Cloning and Expression in Escherichia coli of the Recombinant His-Tagged DNA Polymerases from Pyrococcus furiosus and Pyrococcus woesei
Sawomir 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 containing the structural genes for DNA polymerases of the archaeons Pyrococcus furiosus and Pyrococcus woesei were cloned into an expression vector. The clones expressing thermostable His-tagged DNA polymerases were selected. The cloned fragments were sequenced. The DNA sequences were veried to be authentic by sequencing several clones. The nucleotide (nt) sequence revealed that DNA polymerase of P. woesei (Pwo DNA polymerase) consists of 775 amino acids and has a molecular weight of 90,566. It shows 100% nucleotide identity to the nucleotide sequence of DNA polymerase from P. furiosus (Pfu DNA polymerase). The results conrm that nucleotide sequences of both archaeons (P. furiosus and P. woesei) are highly similar. The recombinant DNA polymerases (His-tagged Pfu and His-tagged Pwo) contained a polyhistidine tag at the N-terminus (43 additional amino acids) that allowed single-step isolation by Ni-afnity chromatography. We found that recombinant plasmids are toxic or unstable in the expressing strain BL21(DE3), even in the absence of the inducing agent, IPTG. However, the plasmids were stable in BL21(DE3) containing the pLysS plasmid, which suppresses expression prior to induction, and His-tagged proteins were expressed upon IPTG addition. The proteins were puried by heat treatment (to denature E. coli proteins), followed by metal-afnity chromatography on Ni2 -Sepharose columns. The enzymes were characterized and displayed high DNA polymerase activity and thermostability. This bacterial expression system appears to be the method of choice for production of Pfu or Pwo DNA polymerases. 1998 Academic Press Key Words: PCR; DNA polymerase; recombinant protein; metal-afnity chromatography; DNA sequence.

1 To whom correspondence should be addressed. E-mail: kur@altis. chem.pg.gda.pl.

A number of thermophilic DNA polymerases have been isolated previously and characterized from both mesophilic eubacteria and archaea sources. Those that have been analyzed are monomeric in solution with molecular masses of 80 115 kDa (13). As expected, these enzymes have elevated temperature optima and have thermal stabilities that roughly correspond to the thermal extremes of the environment from which they were isolated. Despite the fact that thermal stabilities of the native proteins vary among the enzymes, an optimal temperature for polymerization of 70 80C is common. As has been pointed out by many authors, this suggests that the template stability rather than the intrinsic enzyme stability determines the optimal temperature for polymerization (2,3). Due to the thermostability of those enzymes, the structure and function relationships and the potential industrial applications of many thermostable enzymes such as DNA polymerases are of considerable interest to researchers. However, most native thermostable enzymes are synthesized at very low levels by the thermophilic bacteria and are therefore cumbersome to purify. More than 50 DNA polymerase genes have been cloned and sequenced from various organisms, including thermophiles and archaea. Amino acid sequences deduced from their nucleotide sequences can be classied into four major groups: Escherichia coli DNA polymerase I (family A), DNA polymerase II (family B, -like DNA polymerases), DNA polymerase III (family C), and others (family X) (4). In 1975 a new concept for afnity purication of proteins was presented by Porath and co-workers (5). The method is based on the interaction between the side chains of certain amino acids, particularly histidines, on a protein surface and the immobilized transition of metal ions, and is known as immobilized metal ion afnity chromatography (IMAC). The metal ions are immobilized by the use of a chelating agent capable of presenting the metals for
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binding to the protein. Several gene fusion systems employing histidine-rich tags for purication of recombinant proteins have been described so far. The tags, either N- or C-terminal, consisting of consecutive histidine residues binding selectively to immobilized Ni2 ions were described by Houchuli and co-workers (6). The adsorption of the poly-His-tagged proteins to a metal-chelate adsorbent was performed at neutral or slightly alkaline pH, at which the imidazole group of histidine is not protonated (7). The expressed fusion proteins were recovered with purity of more than 90% in a single step using a Ni2 -nitrilotriacetic acid (NTA) adsorbent and elution with low pH or by competition using imidazole (6,7). The use of polyhistidine domains as fusion tags for protein purication has been demonstrated for recombinant fusion proteins produced in a wide range of host cells including E. coli, Saccharomyces cerevisiae, mammalian cells, and baculovirusinfected Spodoptera frugiperda insect cells (8 13). The use of polyhistidines as fusion tags has been demonstrated also for the purication of active Taq and Tth thermostable DNA polymerases (14; Dabrowski and Kur, manuscripts in preparation). An important advantage of His6 afnity tag is the possibility of purifying proteins under denaturing conditions, thus facilitating purication of less soluble proteins. Extremely thermostable DNA polymerases have been puried from some archaeons (13,15) and the genes have been cloned (16,17). The deduced amino acid sequences of the DNA polymerases showed that they all belong to the family. The DNA polymerase from Pyrococcus furiosus (Pfu) has gained considerable attention in the eld of DNA amplication. It is a thermophilic DNA polymerase with 3 -5 exonuclease activity that corrects errors introduced during the polymerization. Several thermophilic DNA polymerases, including Pfu, Vent, Pwo, and UlTma, also have proofreading ability, but they differ in their error rate (18, 21). The error rate for Pfu is reported to be 7- to 10-fold lower than that of nonproofreading Taq DNA polymerase, and 2- to 30-fold lower than other proofreading enzymes (2123). The Pfu DNA polymerase was initially characterized for the preparation isolated directly from P. furiosus (16), but this thermophilic, anaerobic bacterium is difcult to grow so as to obtain large amounts of protein. A major advance was expression of recombinant Pfu DNA polymerase in a baculovirus-mediated system (24). This system makes possible production of commercial amounts, but is not as cheap and convenient as an E. coli bacterial expression system. The Pfu DNA polymerase was also produced in E. coli expression system as a native protein giving milligram quantities of enzyme (25). The results of this study demonstrate that His6tagged Pfu and Pwo DNA polymerases can be efciently synthesized in a biologically active form in the

E. coli overexpression system and that large amounts of active enzyme can be puried using a Ni2 -Sepharose single-step chromatography procedure. We report also the previously unknown nucleotide sequence of the P. woesei DNA polymerase gene and the deduced amino acid sequence of its protein.
MATERIALS AND METHODS

Bacterial Strains, Plasmids, Enzymes, and Reagent Pyrococcus furiosus (DSM 3638) and P. woesei (DSM 3773) strains were obtained from DSMZ-Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH (Braunschweig, Germany) and were used as sources of the total DNA for PCR amplications. The E. coli DH5 strain was used for preparation of plasmids and cloning, and BL21 (DE3) pLysS (Promega, U.S.A.) was applied to expression of His6-tagged Pfu and Pwo DNA polymerases. The plasmid pET30-LIC (Novagen, UK) was used for the construction of the expression system. The E. coli cells with plasmids were cultured aerobically at 37C in LB medium supplemented with a 34 g/ml kanamycin for the DH5 strain or with 50 g/ml chloramphenicol and 34 g/ml kanamycin for the BL21 (DE3) pLysS strain. Restriction and modication enzymes were purchased from Promega. The reagents for PCR and Ni2 -TED Sepharose columns were obtained from DNA-Gdansk (Poland). IPTG, aga rose, and reagents for protein purication were purchased from Sigma. Cloning of the DNA Polymerase Genes from P. furiosus and P. woesei The P. furiosus or P. woesei cells were grown at 95C without shaking, in broth as described for P. furiosus by Uemori et al. (26). DNA was isolated using a Genomic DNA Prep Kit (A&A Biotechnology, Poland). The high homology between the known DNA and amino acid sequences of P. furiosus and P. woesei (Table 1) suggests that both archaeon are very similar. Based on the known DNA sequence of Pfu DNA polymerase gene (GenBank Accession No. D12983) the specic primers for Pfu pol gene were synthesized and used for the amplication from DNA templates of P. furiosus and P. woesei. The PCR conditions were rst designed for the efcient amplication of the Pfu DNA polymerase gene and then applied to the amplication of Pwo DNA polymerase gene. The reaction solution consisted of P. furiosus or P. woesei DNA (100 ng), 2 l (10 M) of each primer (Del1-LIC 5 GAC GAC GAC AAG ATG ATT TTA GAT GTG GAT TAC 3 ; Del2-LIC 5 GAG GAG AAG CCC GTT GAT ATC TAT CGC TTT TCT AGG A 3 ), 5 l (10 mM) dNTPs, 5 l 10 PCR buffer (100 mM Tris-HCl, pH 8.9, 500 mM KCl, 20 mM MgCl2, 1% Triton X-100), and 2 u of thermostable Pfu

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TABLE 1 Identity of Known Nucleotide and Protein Sequences of the Archaebacteria Pyrococcus furiosus and Pyrococcus woesei
Identity of nucleotide sequence (%) 100 100 98.6 100 99.9 Identity of protein sequence (%) 100 100 97 100 99.8

Aligned gene sequences Transcription factor TFIIB TATA-box binding protein Glutamine synthetase glnA DNA polymerase -Galactosidase
a

GenBank Accession No. [P. furiosus (DSM3638)/P. woesei (DSM 3773)] U48391 /X70668 U48392 /U10285 L12410 /X60161 D12983 /U84155a E08095 /AF043283

This study.

DNA polymerase (Stratagene); 30 cycles were performed with a temperature prole of 1 min at 93C, 1 min at 45C, and 2 min at 72C in a Hot-Shot12 thermal cycler (DNA Gdansk, Poland). The amplication products were analyzed by electrophoresis on a 1% agarose gel stained with ethidium bromide. Specic, approximately 2370-bp PCR products were obtained for Pfu DNA polymerase as well as Pwo DNA polymerase gene. Both products gave the same electrophoretic pattern after restriction endonucleases EcoRI, HinfI, HindIII, BamHI, and MspI digestions (data not shown). The PCR products were cloned using the ligaseindependent cloning (LIC) system (Novagen, UK) into pET30-LIC plasmid expression vector (Novagen, UK). First, a PCR product from P. furiosus or P. woesei templates was puried from an agarose gel slice after electrophoresis using Gel Out kit (A&A Biotechnology) and treated with T4 DNA polymerase in the presence of dATP for ligation-independent cloning into the pET30-LIC plasmid expression vector. The plasmids were initially propagated in the E. coli DH5 and the selected clones having recombinant plasmids with large inserts were digested with EcoRI, BamHI, and HindIII, so as to conrm the identity of the cloned DNA fragments. Purication of Recombinant His6-Tagged Pfu or Pwo DNA Polymerase Escherichia coli strain BL21(DE3) pLysS transformed with pET30pfu or pET30pwo was grown at 37C in 1000 ml LB containing 50 g/ml chloramphenicol and 34 g/ml kanamycin to an A660 of 0.3. IPTG was then added to the nal concentration of 1 mM. The cells were harvested after 4 h by centrifugation (9 g of cell mass) and the pellet was resuspended in 20 ml of buffer B (20 mM Tris, pH 7.9, 500 mM NaCl, 5 mM imidazole, 0.1% Triton X-100). The cells were disrupted by sonication, and the insoluble debris was removed by centrifugation. For purication the cleared lysate was immersed in a 75C water orbital shaker and shaken

for 30 min, cooled on ice for 20 min, and then centrifuged at 16,000g at 4C for 20 min. The claried supernatant (about 19 ml) was then applied directly onto a Ni2 -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 the same buffer until the UV absorption returned to the baseline. His6-tagged Pfu/Pwo DNA polymerase was eluted three times with 10 ml of washing buffer B containing 40, 60, and 100 mM imidazole, respectively (buffers B40, B60, and B100). The eluted fractions were pooled and dialyzed against a buffer containing 20 mM Tris, pH 7.9, 100 mM KCl, 0.1% Triton X-100, 0.1 mM EDTA, 1 mM DTT, and 50% glycerol. The concentration of puried enzymes was determined from ultraviolet absorption, using the extinction coefcient for A280 0.78 for 1 mg/ml, calculated from the number of trp and tyr residues in the sequence (using the Protean program of DNAstar, Madison, WI) and also estimated by the Pierce BCA assay using BSA as a standard. The native Pfu or Pwo DNA polymerase enzyme was obtained by proteolytic cleavage of 3 g of recombinant His6-tagged DNA polymerase with 0.05 u of enterokinase (Novagen, UK) in a buffer containing 20 mM Tris-HCl, pH 7.4, 50 mM NaCl, and 2mM CaCl2 at 23C for 16 h. Determination of Pfu and Pwo DNA Polymerases Genes Nucleotide Sequences The independently derived clones expressing thermostable DNA polymerase were selected and sequenced using an ALFexpress DNA sequencer (Pharmacia Biotech, Sweden) using an ALFexpress AutoRead sequencing kit (Pharmacia Biotech, Sweden). Both strands were read entirely and 2370 bases were determined. Assays for Relative DNA Polymerase Activity The relative DNA polymerase activities were determined by comparing band intensities of the PCR-

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

amplied DNA obtained with His6-tagged Pfu, His6tagged Pwo, the native Pfu (Stratagene), and the native Pwo (Boehringer Mannheim) DNA polymerases. The PCR system of bovine leukemia virus (BLV) detection and PCR conditions used in this assay were as described by Kubis et al. (27) with some modications. The primers (ZM4-5 CTC GCC CTC CCG GAC GCC CA and ZM5 - 5 GCT AGG CCT AAG GTC AGG GCC GC) were applied to amplify a 218-bp fragment of the env virus gene. The pENV1 plasmid was used as the matrix DNA. A 1- l portion of plasmid DNA (10 pg) was combined with 2.5 l of 10 reaction buffer (500 mM KCl, 100 mM Tris-HCl, pH 8.8, 25 mM MgCl2, 1% Triton X-100), 2.5 l of a deoxynucleosides triphosphate (dNTP) mixture (2.5 mM of each dNTP), 1 l of each primer (10 M), and 16 l of water. This mixture was supplemented with different volumes of recombinant enzyme fraction (nal preparate of the His6tagged Pfu or Pwo polymerase) or 1 u of the native Pfu or Pwo DNA polymerase. The amplication reactions were performed with an automated thermocycler (thermocycler Hot-Shot 25, DNA-Gdansk) according to the following scheme: an initial cycle of 1 min at 94C was followed by 30 cycles of denaturation for 30 s at 94C, annealing and elongation of primers for 1 min at 64C, and an extension step at 72C for 5 min. PCR-specic products were separated electrophoretically on a 2% agarose gel using 1 Tris-borate EDTA

running buffer at a eld strength of 8 V/cm. The DNA was visualized on an ultraviolet trnsilluminator following ethidium bromide staining and photographed. The Examination of His6-Tagged Pfu or Pwo Polymerase Thermostability For the thermostability studies, His6-tagged Pfu or Pwo polymerase was heat-treated at 98C for 1, 2, 4, 6, and 8 h before PCR reaction. After various incubation periods the enzyme samples (1 unit) were withdrawn and tested for the DNA polymerase activity using specic PCR amplication with pENV1 matrix as described above.
RESULTS AND DISCUSSION

Construction of the Recombinant Vectors Producing His6-Tagged Pfu or Pwo DNA Polymerase The pET30-LIC system is one of the most powerful systems developed for cloning and expression of recombinant proteins in E. coli (28). The pET30-LIC vector has a very strong T7 promoter and can be grown in combination with pLysS to provide additional stringency (29,30). We amplied the pfu or pwo gene using the proofreading Pfu DNA polymerase (Stratagene). The amplication product correspondng to the full-size gene (2370 bp) of P. furiosus or P. woesei DNA poly-

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FIG. 2. SDS electrophoresis in 10% polyacrylamide gel of the fractions obtained by purication of the recombinant His6-tagged Pfu DNA polymerase (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 pETpfu (A) or pETpwo (B); lanes 4, cleared lysate after heat treatment (5 concentrate); lanes 5 and 6, owthrough fraction with the B buffer; lanes 7, washed fraction with the B20 buffer; lanes 8, eluted fraction with the B40 buffer; lanes 9, eluted fraction with the B60 buffer; lanes 10, eluted fraction with the B100 buffer.

merase was treated with T4 DNA polymerase in the presence of dATP so as to create LIC ends, and inserted into pET30-LIC vector, thus obtaining the recombinant pET30pfu or pET30pwo plasmid. The nucleotide sequences of these constructs were conrmed by sequencing analysis to ensure that the reading frame is correct. The primer, nucleotide, and amino acid sequences of the pETpfu or pETpwo plasmid near the site of the DNA polymerase gene insertion are shown in Fig. 1. The obtained genetic constructs retained the open reading frame, and the target proteins contained 43 additional amino acid residues at N-terminus, including a cluster of six histidine residues for purication of the recombinant proteins by metal-afnity chromatography. Determination of DNA Pwo pol Gene Sequence All clones of pET30pwo exhibited the same nucleotide sequence of pol gene as was determined by sequencing using an ALFexpress DNA sequencer and an ALFexpress AutoRead sequencing kit (Pharmacia Biotech). The alignment analyses of the obtained DNA Pwo pol gene sequence and known DNA Pfu pol gene sequence performed with NALIGN program (PCGENE package) revealed 100% identity. The results of our sequencing data conrm that P. furiosus and P. woesei are closely related organisms and were comparable to the identities obtained in the alignments within the group of known sequences for P. furiosus and P. woesei (see Table 1). The complete sequence of P. woesei DNA polymerase gene can be obtained from GenBank (Accession No. U84155).

Expression of the Recombinant His6-Tagged Pfu or Pwo DNA Polymerases The level of expression was examined in different strains. When pETpfu or pETpwo was transformed into E. coli BL21(DE3) at 37C no colonies were obtained. This observation suggested that pETpfu or pETpwo plasmide might produce small amounts of recombinant polymerase prior to IPTG induction with toxic effect to E. coli. The recombinant plasmid pET30pfu or pET30pwo was then transformed into E. coli BL21(DE3) pLysS. The pLysS plasmid, which expresses T7 lysozyme in the bacterial cytoplasm, strongly represses protein expression from the pET vectors in the absence of induction, thus

FIG. 3. Monitoring of the recombinant His6-tagged Pfu DNA polymerase activity (A) and His6-tagged Pwo DNA polymerase activity (B) of the fractions obtained during purication procedures using specic PCR reaction. Lanes 1 represent the molecular weight marker (501, 489, 404, 331, 242, 190, 147, 111, 110 bp); lanes 2, 0.1 l of the claried supernatant after heat treatment; lanes 3 and 4, 1 l of the owthrough fraction with the B buffer; lanes 5, 1 l of the fraction washed with the B20 buffer (20 mM imidazole); lanes 6, 1 l of the fraction eluted with the B40 buffer (40 mM imidazole); lanes 7, 1 l of the fraction eluted with the B60 buffer (60 mM imidazole); lanes 8, 1 l of the fraction eluted with the B100 buffer (100 mM imidazole).

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TABLE 2 Purication of His6-Tagged Pfu or Pwo DNA Polymerase Expressed in E. coli

Total protein His6-tagged Pfu ND 60.0 24.0
a

(mg)

Specic activity (units/mg) His6-tagged Pfu ND ND 31,000 His6-tagged Pwo ND ND 31,000

Purity (%)b His6-tagged Pfu 6 40 97 His6-tagged Pwo 7 42 97

Purication step Total bacterial lysatec Heat-soluble proteins Metal-afnity chromatography

His6-tagged Pwo ND 63.3 26.6

a The concentration of heat-soluble proteins was determined by the Pierce BCA assay using BSA as a standard. The concentration of protein after metal afnity chromatography was determined from ultraviolet absorption, using the extinction coefcient for A280 0.78 for 1 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 SDSPAGE gel stained with Coomassie blue using Biometra ScanPack software. ND, not determined. c Nine grams of the wet weight of cells used in the preparation.

enabling expression of very toxic proteins. The mechanism of the additional stringency is that the T7 lysozyme binds to and inactivates T7 RNA polymerase (which is expressed at low levels in the absence of induction) and inhibits transcription. Following induction, the amount of T7 RNA polymerase is sufcient to overcome the lysozyme inhibition. We found that colonies could easily be obtained at 37C when pETpfu or pETpwo was used to transform the E. coli BL21(DE3) carrying pLysS plasmid. Bacterial colonies grown on chloramphenicol and kanamycin-containing agar were assayed for DNA polymerase activity using specic PCR amplication with the pENV1 matrix. The clones displaying high DNA polymerase activity were selected. The selected clone was grown in LB medium supplemented with the chloramphenicol and kanamycin. Overexpression was induced by IPTG addition (nal concentration, 1 mM) at various stages of culture (A660: 0.1, 0.2, 0.3, 0.4, 0.5, and 0.6). The best induction of His6-tagged Pfu or Pwo DNA polymerase synthesis was obtained at A660 of 0.3. At A660 0.4 the synthesis of the recombinant protein and DNA polymerase activity were signicantly lower. Purication of the Recombinant His6-Tagged Pfu or Pwo DNA Polymerases The purity of enzymes was examined by sodium dodecyl sulfatepolyacrylamide gel electrophoresis (SDS PAGE) (Fig. 2). Monitoring of the His6-tagged Pfu or Pwo DNA polymerase activity was carried out by PCRspecic reaction with 1 l of protein fractions withdrawn during purication procedure (Fig. 3). His6tagged Pfu or Pwo DNA polymerases were expressed as a soluble form in the cytosol. Before the chromatography step, the cell lysate was heat treated at 75C for 30 min. The heat denaturation step resulted in the precipitation of the vast majority of contaminating cellular proteins (Figs. 2A and 2B, lanes 4). Several E. coli proteins still remained soluble after the heating step.

However, at this stage, His6-tagged Pfu or Pwo DNA polymerases were pure enough for PCR applications. For the nal purication, the claried medium was chromatographed on a Ni2 -TED Sepharose column. The recombinant enzymes were eluted from the column as a single peak (about 97% purity) (Figs. 2A and 2B, lanes 6 8). A band corresponding to a 95-kDa protein was observed in SDSPAGE of crude extracts of E. coli BL21(DE3) pLysS pET30pfu or E. coli BL21 (DE3) pLysS pET30pwo cultures after IPTG induction (Figs. 2A and 2B, lanes 3). This band was absent in the control crude extracts of E. coli BL21 (DE3) pLysS cultures (Figs. 2A and 2B, lanes 2). The pronounced enrichment of the 95-kDa protein depicted in Fig. 2 (lanes 4) after the heat denaturation step (compare lane 4 with lane 3 in Figs. 2A and 2B) should be noted. The amount of the 95-kDa protein, and consequently the specic activity, was estimated by ultraviolet absorption (A280) determination or using BSA as a standard (see Table 2). We obtained 24 mg of puried His6-tagged Pfu polymerase or 26.6 mg of puried His6-tagged Pwo, as determined by the A280. After the

FIG. 4. The DNA polymerase activity of the His6-tagged Pwo DNA polymerase after purication by afnity chromatography on the Ni2 -TED Sepharose (DNA-Gdansk) determined using specic PCR amplication. Lane 1, the molecular weight marker (501, 489, 404, 331, 242, 190, 147, 111, 110 bp); lanes 27, puried enzyme of 2, 1, 0.5, 0.3, 0.2, and 0.1 l, respectively; lane 8, 1 u of the commercial Pwo DNA polymerase (Boerhringer Mannheim).

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FIG. 5. Temperature stability assay. The His6-tagged Pwo DNA polymerase (1 u, 0.032 g) was heat treated at 98C before PCR reaction for 0 h (lane 2), 1 h (lane 3), 2 h (lane 4), 4 h (lane 5), 6 h (lane 6), 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.

dialysis of recombinant proteins against storage buffer, the DNA polymerase activity was 2.8 units/ l (i.e., 746,000 units/liter medium, 31,000 units/mg) for the puried His6-tagged Pfu DNA polymerase and 3.1 units/ l (i.e., 827,000 units/liter medium, 31,000 units/ mg) for the puried His6-tagged Pwo DNA polymerase isolated from 1-liter E. coli cultures (see also Table 2). We have found that the reproducible high yields of His6-tagged enzymes are obtained from freshly transformed cells, whereas the expression from a freezer stock gave erratic and lower yields. Examination of the Relative DNA Polymerase Activity The relative DNA polymerase activity was determined by comparing the PCR results obtained for the His6-tagged Pfu or Pwo DNA polymerase and the native Pfu DNA polymerase (Stratagene) (results not shown) or the native Pwo DNA polymerase (Boehringer Mannheim) (Fig. 4) using specic PCR amplication of the BLV env gene target. As shown in Fig. 4, the relative His6-tagged Pwo DNA polymerase activity was about 3.1 u/ l and about 2.8 u/ l for His6-tagged Pfu DNA polymerase (results not shown). The relative His6-tagged Pfu or Pwo DNA polymerase activity was also comparable to the His6-tagged Pfu or Pwo DNA polymerase cleaved with enterokinase (result not shown) using the same specic PCR amplication test. Thermostability His6-tagged Pfu or Pwo DNA polymerase was very stable and active at high temperature. The thermal inactivation level at 98C of the puried His6-tagged Pwo enzyme is shown in Fig. 5.
CONCLUSIONS

from native sources or that expressed in the baculovirus system in case of Pfu DNA polymerase. 2. His6-tagged Pfu or Pwo DNA polymerase was puried in a single chromatography step using a Ni2 TED Sepharose column (97% purity). 3. The 43 additional N-terminal amino acid residues (including a cluster of six histidine residues for purication of the recombinant protein by metalafnity chromatography) have no effect on polymerase activity. 4. The effective cloning of the PCR products obtained using His6-tagged Pfu or Pwo DNA polymerase into blunt-ended vectors suggested the presence of 3 -5 exonuclease activity in the recombinant proteins. 5. The applied overexpression system was very efcient, giving 31,000 units/mg of the puried His6tagged Pfu or His6-tagged Pwo DNA polymerase activity. This is similar to the activity reported for Pfu directly puried 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 puried recombinant enzymes exhibited high polymerase activity and high thermostability.
ACKNOWLEDGMENTS
The work was supported by the Technical University of Gdansk and DNA-Gdansk II s.c.

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

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