GBF (Gesellschaft für Biotechnologische Forschung), Department of Structure Research, Mascheroder Weg 1, D-38124 Braunschweig and 2 Roche Molecular Biochemicals, Nonnenwald 2, D-82377 Penzberg, Germany
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Abstract |
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Keywords: chimeric DNA polymerase/protein design/structure/function relationship/Taq DNA polymerase/thermostability
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Introduction |
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The determination of the three-dimensional (3D) structure has shown that, like the E.coli pol I, Taq polymerase has a 5'3' exonuclease domain at its amino terminus and a C-terminal polymerase domain. The E.coli pol I supports a 3'5' exonuclease proof-reading activity in the intervening domain, whereas the corresponding domain in the Taq polymerase has no catalytic function. A comparison of the 3D structures showed that the polymerase domains of the Taq polymerase and the Klenow fragment of the E.coli pol I are nearly identical, whereas the intervening domains differ extensively (Kim et al., 1995; Korolev et al., 1995
). From the structure of the Tne polymerase, only the amino acid sequence is known at present (Roche Molecular Biochemicals). The enzyme supports 5'3' exonuclease, 3'5' exonuclease and polymerase activity and is homologue to the E.coli pol I, which allows structure prediction by homology without knowing the experimental three-dimensional structure.
The mesophile E.coli pol I and its Klenow fragment (Jacobsen, 1974; Kornberg and Baker, 1992
) were used in the PCR reaction before the introduction of thermostable DNA polymerases. These enzymes are less useful in routine PCR application, as they are heat labile, which means that fresh aliquots of the enzyme have to be added at each PCR cycle.
The Tne polymerase was isolated from the thermophilic eubacteria Thermotoga neapolitana and shows, like the Taq polymerase, high thermostability. The amino acid sequence is similar to the Thermotoga maritima DNA polymerase (UlTma polymerase) (B.Frey, unpublished work, 1995). For some applications, e.g. long PCR, the low polymerization rate compared with the Taq polymerase is disadvantageous. Proof-reading DNA polymerases are used in PCR when high accuracy is required.
The ability to engineer hybrid enzymes, which contain elements of two or more enzymes, have been shown to be useful to generate proteins with new properties by alteration of non-enzymatic and enzymatic properties and as tools for understanding structurefunction relationships (reviewed in Nixon et al., 1998). Investigations on DNA polymerases have been described by Tabor and Richardson (1995). They showed that the construction of active site hybrids and the introduction of mutations increase the discrimination with respect to the four dideoxynucleotides. These DNA polymerases have enhanced properties for use in DNA sequence analysis. For high-fidelity PCR, today often thermostable DNA polymerases with 3'5' exonuclease proof-reading function are used, such as Vent DNA polymerase (Mattila et al., 1991) or Pfu DNA polymerase (Lundberg et al., 1991
; Cline et al., 1996
). Recently, mixtures of non-proof-reading, e.g. Taq polymerase, and proof-reading DNA polymerases, e.g. Pfu or Vent DNA polymerase, were used to synthesize higher yields of PCR product, allowing amplification of longer templates (Barnes, 1994
). Several investigations have been described to enhance the fidelity of the Taq polymerase. Thus, the deletion of the 5'3' exonuclease domain resulted in an enzyme with reduced tendency toward errors (Barnes, 1992
) and substitutions in the polymerase domain increased the efficiency of the synthesis of long DNA molecules (Ignatov et al., 1998
). Furthermore, Park et al. (1997) described a slight increase in 3'5' exonuclease activity of the Taq polymerase by protein engineering in the active site.
Another method to create novel enzymes which catalyse particular reactions is the building-block approach, where functional domains serve as building blocks that can be exchanged (Nixon et al., 1998). The Taq polymerase, the E.coli pol I and presumably the Tne polymerase consist of structural and functional independent domains. In order to test the hypothesis that a 3'5' exonuclease function can be introduced into the Taq polymerase by domain exchange, we cloned three chimeric DNA polymerases. The chimera TaqEc1, where the intervening domain of the Taq polymerase was exchanged for the 3'5' exonuclease domain of the E.coli pol I, was designed using the 3D structure of the Klenow fragment of the E.coli pol I and a 3D model of the two C-terminal domains of the Taq polymerase. The chimeras TaqTne1 and TaqTne2 I were designed using a 3D model of the two C-terminal domains of the Taq polymerase and a 3D model of the two C-terminal domains of the Tne polymerase. In the chimera TaqTne1 the intervening domain of the Taq polymerase was exchanged for the 3'5' exonuclease domain of the Tne polymerase. Villbrandt et al. (1997) showed that the polymerase domain of the Taq polymerase is destabilized by removing the adjacent domain. In order to obtain more information about the importance of the interactions at the interface between the two domains with respect to function and thermostability in thermostable DNA polymerases, we cloned the chimera TaqTne2. Here, additionally, the residues, that form the N-terminal
-helix of the polymerase domain of the Taq polymerase were exchanged for the corresponding residues of the Tne polymerase, which means that the interface of the two domains contains the network of interactions from the Tne polymerase. The N-terminal his-tagged clones were overproduced and were purified using a single-step isolation by Ni2+ affinity chromatography. The enzymatic properties were examined in vitro.
At the beginning of the project only the primary sequences of DNA polymerases from a wide range of organisms (Braithwaite and Ito, 1993), the primary sequence of the Tne polymerase (Roche Molecular Biochemicals), the coordinates of the Klenow fragment including the 3'5' exonuclease and the polymerase domain of the E.coli pol I (Beese et al. 1993
) and a stereo picture of the Taq polymerase (Kim et al., 1995
) were available. Later, the X-ray coordinates of the Taq polymerase became available from the protein structure data bank. They were used to evaluate the reliability of our model.
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Materials and methods |
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Amino acid alignments were performed using the program GCG (Devereux et al., 1984) and the following coordinates were taken from the Brookhaven Protein Data Bank: Klenow fragment of the E.coli pol I (Beese et al., 1993; PDB code 1KLN), Taq polymerase (Kim et al., 1995; PDB code 1TAQ).
C coordinates were picked using the program Magick (J.Cristy, E. I. du Pont De Nemours) and z-coordinates were calculated with the program Stereo (Collaborative Computational Project, 1994
). For the generation of protein backbone atoms from C
coordinates the program O (Jones et al., 1991
) was used.
The molecular modelling, the r.m.s. deviation and the r.m.s. fitting based on C distance comparison were carried out using the program BRAGI, version 5.0 for Hewlett-Packard and Silicon Graphics Workstations (Schomburg and Reichelt, 1988
; Lessel and Schomburg, 1994
) as described in Villbrandt et al. (1997).
Force field calculations were performed with the program AMBER, VAX version 3.0 (Weiner, 1984) as described in Villbrandt et al. (1997). The quality of model structures was checked using the program Procheck (Laskowski et al., 1993). Calculations were performed on a Hewlett-Packard graphic workstation and a DEC alpha workstation. The presentation of the superposition of structures (Figure 1a and b
) was created using the program GRASP, version 1.3 (Nicholls et al., 1991
).
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Plasmids containing the genes of the Taq polymerase, the E.coli pol I and the Tne polymerase as well as the plasmid pa were kindly provided by Dr B.Frey (Department of Molecular Biology, Roche Molecular Biochemicals, Penzberg, Germany). The plasmid Pbtaq containing the gene of the Taq polymerase was used in the modified form described in Villbrandt (1995). Non-modified oligonucleotides were synthesized by Life (Gibco-BRL) and Dig-labeled oligonucleotides were synthesized by TIB MOLBIOL. For mutagenesis, cloning and expression of the genes, the E.coli strain XL1-Blue (Bullock et al., 1987) was used.
Gene manipulation and gene expression
Standard molecular biological techniques were carried out as described in Sambrook et al. (1989). Primer sequences are given in Table I. For the mutagenesis and expression of the his-tagged Taq polymerases the modified plasmid Pbtaq was used, where the genes were expressed after induction with isopropyl-ß-D-thiogalactopyranoside (IPTG) as soluble proteins.
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For the generation of the hybrid genes for the chimeric DNA polymerases the SOE (splicing by overlap extension) method (Horton et al., 1989) was used. The construction of each hybrid gene included five PCR reactions. The modified plasmid Pbtaq containing the gene of the N-terminal his-tagged Taq polymerase was used as template in each PCR reaction 1 and 3. For the chimera TaqEc1 the PCR 1 was performed using the oligonucleotides NHis-oligo1 and Ec-oligo2 to produce the fragment Ec-F1; PCR 2 using the plasmid containing the gene of the E.coli pol I, the oligonucleotides Ec-oligo3 and Ec-oligo4 to produce the fragment Ec-F2; PCR 3 using the oligonucleotides Ec-oligo5 and NHis-oligo2 to produce the fragment Ec-F3; PCR 4 using the fragments Ec-F1, Ec-F2 and the oligonucleotides NHis-oligo1 and Ec-oligo4 to produce the fragment Ec-F4; PCR 5 using the fragments Ec-F3, Ec-F4 and the oligonucleotides NHis-oligo1 and NHis-oligo2 to produce the fragment Ec-F5.
For the chimera TaqTne1 the PCR 1 was performed using the oligonucleotides NHis-oligo1 and Tne-oligo2 to produce the fragment Tne1-F1; PCR 2 using the plasmid containing the gene of the Tne polymerase, the oligonucleotides Tne-oligo3 and Tne-oligo4 to produce the fragment Tne1-F2; PCR 3 using the oligonucleotides Tne-oligo5 and NHis-oligo2 to produce the fragment Tne1-F3; PCR 4 using the fragments Tne1-F1, Tne1-F2 and the oligonucleotides NHis-oligo1 and Tne-oligo4 to produce the fragment Tne1-F4; PCR 5 using the fragments Tne1-F3, Tne1-F4 and the oligonucleotides NHis-oligo1 and NHis-oligo2 to produce the fragment Tne1-F5.
For the chimera TaqTne2 the PCR 1 was performed as described for the variant TaqTne1; PCR 2 using the plasmid containing the gene of the Tne polymerase, the oligonucleotides Tne-oligo3 and Tne2-oligo4 to produce the fragment Tne2-F2; PCR 3 using the oligonucleotides Tne2-oligo5 and NHis-oligo2 to produce the fragment Tne2-F3; PCR 4 using the fragments Tne2-F1, Tne2-F2 and the oligonucleotides NHis-oligo1 and Tne2-oligo4 to produce the fragment Tne2-F4; PCR 5 using the fragments Tne2-F3, Tne2-F4 and the oligonucleotides NHis-oligo1 and NHis-oligo2 to produce the fragment Tne2-F5.
The final PCR fragments NHis-F, 5DHis-F3, Ec-F5, Tne1-F5 and Tne2-F5 were EcoRI/PstI-digested and cloned into the plasmid Pbtaq. Subsequently the DNA fragments were separated on a agarose gel and isolated using the QIAquick gel extraction kit (Qiagen). Ligation of DNA fragments and transformation of E.coli by electroporation was carried out as described in Villbrandt (1995). Clones were picked and plasmid-DNA was isolated using the QIAprep Spin plasmid kit (Qiagen). Gene expression was induced in 1 l of LB medium containing 100 µg/ml ampicillin, 12.5 µg/ml tetracycline and 1 mM IPTG for 16 h at 37°C.
DNA sequencing
Mutated regions of the genes were sequenced on both DNA stands. The genes encoding the his-tagged Taq polymerases were sequenced with an ALF sequencer and an AutoRead sequencing kit (Pharmacia) using the protocol and reagents supplied by the manufacturer. The modified sequences of the genes from the chimeric DNA polymerases were determined by Dr W.Metzger with SEQUISERVE using an Applied Biosystems Model 373A system.
Enzyme purification
Frozen cells were harvested by centrifugation at 6000 g, suspended in 20 ml of lysis buffer (50 mM TrisHCl, pH 8.5, 10 mM 2-mercaptoethanol, 1 mM PMSF) and sonicated for 10 min in an ice bath. Cell fragments were removed by centrifugation and the supernatant was subjected on a nickel-nitrilotriacetic acid (Ni-NTA) agarose (Qiagen) column (1.3x2.7 cm) equilibrated with buffer A (20 mM TrisHCl, pH 8.5, 100 mM KCl, 20 mM imidazole, 10 mM 2-mercaptoethanol, 10% glycerol). Washing steps were performed with 40 ml of buffer A, 10 ml of buffer B (20 mM TrisHCl, pH 8.5, 1 M KCl, 10 mM 2-mercaptoethanol, 10% glycerol) and 10 ml of buffer A. Proteins were eluted with buffer C (20 mM TrisHCl, pH 8.5, 100 mM KCl, 100 mM imidazole, 10 mM 2-mercaptoethanol, 10% glycerol). Fractions of 10 ml (wash fractions) and 1 ml (elution fractions) were collected at a flow-rate of 0.5 ml/min. The fractions were pooled, dialysed against storage buffer (20 mM TrisHCl, pH 8.0, 100 mM KCl, 0.1 mM EDTA, 1 mM DTT, 200 µg/ml gelatine, 0.5% Tween 20, 0.5% Nonidet P40, 50% glycerol) and stored at 20°C for enzyme activity assays. The his tag of the N-terminal his-tagged Taq polymerase was removed using the protease enterokinase and the cleavage mixture was treated on an Ni-NTA agarose column. The purification was carried out as described above. The purity of the proteins was estimated by SDSPAGE on 8% Midget gels (LKB Midget System; Schrägger and von Jagov, 1987) stained with Coomassie Brilliant Blue and on 1015% gradient SDS gels stained with silver on a Pharmacia Phast system. The protein concentration was determined by UV measurement (OD280) and by using the Protein Assay ESL (Roche Molecular Biochemicals).
N-Terminal amino acid sequence analysis
Purified protein samples of the N-terminal his-tagged Taq polymerase were subjected to N-terminal amino acid sequence analysis as described in Villbrandt et al. (1997).
Tests for contaminating activities
The absence of contaminating activities was investigated in 50 µl of buffer (10 mM TrisHCl, pH 7.5, 10 mM MgCl2, 1 mM DTE). Purified enzyme fractions (1, 5, 10 and 20 µl) were incubated with the respective nucleic acids for 16 h at 65°C. Endonucleases were tested using 1 µg of pBR322 and double-stranded nucleases by using 1 µg of the EcoRI/HindIII fragment of lambda DNA. The probes were electrophoresed on 1% agarose gel and stained with ethidium bromide.
Polymerase activity, temperature optimum and thermostability assay
Polymerase activity was assayed with a non-radioactive test system. The test was performed in 50 µl of a mixture containing 5 µl of buffer mix [500 mM TrisHCl, 150 mM (NH4)2SO4, 100 mM KCl, 70 mM MgCl2, 100 mM 2-mercaptoethanol, pH 8.5], 100 µM each dATP, dCTP, dGTP, dTTP, 36 nM digoxigenin-11 dUTP (Roche Molecular Biochemicals), 12 µg of activated calf thymus DNA (Sigma), 10 µg of BSA and 2 µl of chimeric polymerase or N-terminal his-tagged Taq polymerase or 0.02 units of commercial Taq polymerase (Roche Molecular Biochemicals) in storage buffer (see above). A 5 µl volume of each reaction solution was transferred into a white 0.45 µm Biodyne B membrane bottomed well (Pall BioSupport, SM045BWP) and incubated at 70°C for 10 min. The membrane of the plate was treated using a silent monitor vacuum manifold (Pall BioSupport) as follows: incubation (2x) for 2 min with 100 µl of buffer 1 (1% blocking reagent (Roche Molecular Biochemicals) in 0.1 M maleic acid, 0.15 M NaCl, pH 7.5); incubation (2x) for 2 min with 100 µl of buffer 2 (1:10 000 diluted anti-Dig-AP-Fabfragment antibodies (Roche Molecular Biochemicals) in buffer 1); wash step (2x) with 200 µl of buffer 3 (buffer 1 with 0.3% Tween 20); wash step with 200 µl of buffer 4 (0.1 M TrisHCl, 0.1 M NaCl, 50 mM MgCl2, pH 9.5); incubation for 5 min with 50 µl of buffer 5 [1:100 diluted CSPD (Roche Molecular Biochemicals) in buffer 4]. The probes were measured in a luminometer (Microlumar LB 96P, Berthold or Wallac Micro Beta Trilux). In order to calculate enzyme units, the radioactive test system described in Villbrandt et al. (1997) was used to measure the incorporation of [32P]dCTP in M13 mp9 ss-DNA for several probes. In both test systems, each concentration was tested at least twice. For the determination of the temperature optimum of the purified enzymes, the tests were assayed at 25, 37, 50, 60, 72 and 80°C. Thermostability was assayed by incubating the test mixtures at 80 or 95°C for 1, 3 or 5 min. Samples were chilled on ice and the remaining activities were determined in the non-radioactive test system at 72°C.
DNA amplification
PCR was performed using the AmpliTaq DNA polymerase amplification kit (Perkin-Elmer Cetus) replacing the PCR buffer with the Taq polymerase PCR buffer from Roche Molecular Biochemicals. Two different PCR reactions were performed generating a 500 bp fragment of lambda-DNA using a lambda forward primer and a lambda reverse primer and a 250 bp fragment of plasmid pa using a plasmid pa forward primer and a plasmid pa reverse primer. The 100 µl reaction mixtures contained 1 ng of lambda-DNA or plasmid pa, primers (25 mer) each at 1 µM, 200 µM dNTPs, 10 µl of PCR buffer and 0.5 µl of the chimeric polymerases. PCR was performed for 25 cycles at a 72°C extension temperature (1 min at 94°C, 0.5 min at 50°C, 1 min at 72°C with 2 min template melting at 94°C at the beginning and 7 min extension at 72°C after the last cycle) using 1.25 units of TaqEc1, 3.6 units of TaqTne1 and 3.5 units of TaqTne2 per cycle and at a 55°C extension temperature (1 min at 95°C, 0.5 min at 50°C, 1 min at 55°C with 2 min template melting at 95°C at the beginning and 7 min extension at 55°C after the last cycle) using 6 units of TaqEc1 and 7.5 units of TaqTne1 per cycle. Fresh aliquots of the chimeric polymerases were added in each cycle at 50°C. PCR reactions with the commercial Taq polymerase (Roche Molecular Biochemicals) and the his-tagged Taq polymerases were performed for 25 cycles at a 72°C extension temperature as described above by adding enzyme fractions only at the beginning of the PCR.
3'5' Exonuclease assay
3'5' Exonuclease was assayed using the oligonucleotides P1 [5'-Dig-GGA TCC CAT TGC CCA GGG AAT TC-3'] (matched primer), P2 [5'-Dig-GGA TCC CAT TGC CCA GGG AAT TT-3'] (mismatched primer) and P3 [5'-Dig-GGA TCC CAT TGC CCA GGG AAT TG-3'] (mismatched primer) annealed to the oligonucleotide M1 [5'-CAC TAA AGT AAC TTT ATA AAA TCA AAA GAA TTC CCT GGG CAA TGG GAT CC-3'] (matrix). Reactions were assayed in 10 µl of a mixture containing 1 µl of buffer (100 mM TrisHCl, 15 mM MgCl2, 500 mM KCl, 0.1 mg/ml gelatine, pH 8.3) or polymerase specific buffer for the UlTma polymerase, 1 µl of polymerase, 1 pmol of matrix and 500 fmol of primer P1, P2 or P3. Incubation was performed at different temperatures and different incubation times. DNA fragments were separated on a 12.5% acrylamide gel (SequaGel-Kit, Medco) and blotted on a nylon membrane (Roche Molecular Biochemicals). The membrane was incubated for 30 min in 100 ml of buffer 1 (see above) and 30 min in 100 ml of buffer 2 (see above), washed three times in 135 ml of buffer 3 (see above), incubated for 5 min in 50 ml of buffer 4 (see above) and incubated for 5 min in 50 ml of buffer 5 [1:1000 diluted CPD-Star (Roche Molecular Biochemicals) in buffer 4]. The nylon membrane was dried and exposed on a chemiluminescence film (Roche Molecular Biochemicals) for 3060 min.
3'-Mismatch primer correction assay
Reactions were assayed as described for the 3'5' exonuclease assay but using 20 µl of mixture and additionally 10 µM dATP, dCTP, dGTP and dTTP. Incubation was performed at different temperatures and different incubation times and samples were heated to 95°C for 5 min. Volumes of 10 µl of each mixture were digested with EcoRI. DNA fragments were separated on a 12.5% acrylamide gel, blotted on a nylon membrane and exposed on a chemiluminescence film as described above.
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Results and discussion |
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Although the three-dimensional structure of the Taq polymerase has been known since 1995 (Kim et al., 1995), the molecule coordinates were not available from the protein structure data bank until December 1996. Therefore, at the beginning of the project the structure of the Klenow fragment of the E.coli pol I (Beese et al., 1993, PDB-code:1kln), the stereo picture of the Taq polymerase (Figure 2c
in Kim et al., 1995) and the amino acid sequence of the Tne polymerase (Roche Molecular Biochemicals) served as starting points in developing structural models of the two C-terminal domains of the Taq polymerase and the Tne polymerase.
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As the 3D structures of the intervening domain of the Taq polymerase and the 3'5' exonuclease domain of the Klenow fragment differ extensively (Kim et al., 1995), the intervening domain of the Taq polymerase (amino acids 292423) was reconstructed from a stereo picture of the structure (mentioned above). The picture was scanned, C
coordinates were picked and z-coordinates were calculated. A 3D model consisting of a polyalanine chain (glycines were not replaced) was built using the program O. The site chains were added and the model was refined by force field calculations using constraints to keep the C
coordinates fixed. The two partial models were connected (amino acids 424832 from the model mentioned above) using the structure based alignment of the Taq polymerase and the Klenow fragment (Kim et al., 1995
) produced by the program BRAGI. The whole model structure was refined by force field calculations.
Later, the model structure of the two C-terminal domains of the Taq polymerase could be matched on the corresponding atoms of the X-ray structure (Kim et al., 1995) to give an r.m.s. difference of 1.121 Å in the C
coordinates, 2.511 Å for main chain atoms and 3.393 Å for all atoms. With regard to the intervening domain, the r.m.s. deviation of the model structure from the X-ray structure was 1.001 Å in the C
coordinates, 1.540 Å for main chain atoms and 2.683 Å for all atoms. According to Joyce and Steitz (1994) the polymerase domain can be divided into three subdomains, named `palm', `finger' and `thumb'. The differences between the model structure and the experimental structure are observed mainly in the finger and thumb subdomains. In the palm subdomain, which contains the catalytic centre, the structures are nearly identical. Correspondingly, Korolev et al. (1995) pointed out differences in ordering of the structures between the Klenow fragment and the Klentaq1 in the finger tip and the thumb regions. For these reasons, the result is not surprising, as the model structure of the polymerase domain was built in homology with the structure of the Klenow fragment. Corresponding to Villbrandt et al. (1997), in summary, our model agrees well with the X-ray structure.
A model of the Tne polymerase, which includes residues 297893, was built in homology with the structure of the Klenow fragment and the model structure was refined by force field calculations. All highly conserved regions and catalytically essential amino acids described for the 5'3' exonuclease and polymerase domains of pol I-like DNA polymerases (reviewed in Joyce and Steitz, 1994) are very similar in Taq polymerase, E.coli pol I and Tne polymerase. Additional, the 3'5' exonuclease domains of the E.coli pol I and the Tne polymerase are homologous (Table II). For these reasons, the 3D structure of the Klenow fragment seemed to be suitable for the structure prediction of the Tne polymerase. Like the Taq polymerase, the Tne polymerase is thermostable and seemed to be suitable for the construction of thermostable chimeras.
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Design, expression and purification of the chimeras
For the design of chimeric DNA polymerases, the amino acid sequences of the Taq polymerase, the Tne polymerase and the E.coli pol I were aligned domainwise (data not shown), taking into account known conserved regions, essential amino acids, multiple amino acid alignments of DNA polymerases and known secondary structures and structure-based alignments (reviewed in Joyce and Steitz; 1994). Sequence identities are given in Table II.
The chimera TaqEc1 was designed using an alignment of the structure of the Klenow fragment of the E.coli pol I and the model structure of the Taq polymerase. The whole intervening domain of the Taq polymerase (residues 292423) has been exchanged for the whole 3'5' exonuclease domain of the E.coli pol I (residues 327519) (Figure 1a). The chimeras TaqTne1 and TaqTne2 were designed using an alignment of the structure models of the Taq polymerase and the Tne polymerase. The chimera TaqTne1 contains the whole 3'5' exonuclease domain of the Tne polymerase (residues 295485) instead of the whole intervening domain of the Taq polymerase (residues 292423) (Figure 1b
). In the chimera TaqTne2 the amino acids that form the N-terminal
-helix of the polymerase domain (424448) were also substituted by the corresponding amino acids of the Tne polymerase (486510) to preserve the network of interactions at the interface of the two domains (Figure 1b
).
In order to simplify the purification of the chimeric DNA polymerases, metal affinity chromatography on Ni-NTA agarose was used. For this purpose, two approaches were tested by constructing an N-terminal and an internal his-tagged Taq polymerase. The N-terminal his-tagged Taq polymerases was generated by PCR. The inserted amino acid sequence MRGSHHHHHHAADDDDK contains the epitope [MRGS(H)6] and provides the possibility of detecting the protein using MRGS 'His antibodies (Quiagen) in a Western blot (not tested). Additionally, the his tag can be removed using the protease enterokinase [cleavage site (D)4KX], which has been shown in a pilot experiment (data not shown). The internal his-tagged Taq polymerase was generated by PCR mutagenesis. Six histidines were inserted into a flexible loop of the 5'3' exonuclease domain of the Taq polymerase between glycines 79 and 80. The DNA sequences of both his-tagged Taq polymerases were confirmed by DNA sequencing and the 20 N-terminal amino acids of the N-terminal his-tagged Taq polymerase were confirmed by protein sequencing. Both his-tagged Taq polymerases were expressed at high levels in E.coli as soluble proteins using the plasmid Pbtaq and yielded large amounts of purified protein products. A total of 2.5 mg of protein with 98% purity of the N-terminal his-tagged Taq polymerase and a total of 3.5 mg of protein with 60% purity of the internal his-tagged Taq polymerase were isolated from 1 l of bacterial culture. We assume that the internal his tag is less accessible to affinity material than the N-terminal his tag. Smirnov et al. (1995) described the purification of a Taq polymerase containing 11 additional amino acid residues at the N-terminus, of which six are histidines, on Ni-NTA agarose. Compared with this system, we obtained a higher yield on highly purified N-terminal his-tagged Taq polymerase (2.5 mg compared with 0.81 mg from 1 l of bacterial culture). Furthermore, our system allows the purification of variant enzymes with decreased thermostability, because no heat precipitation is involved. As the purification of the N-terminal his-tagged Taq polymerase resulted in more highly purified protein products than the internal his-tagged Taq polymerase, this construct was used for the cloning, expression and purification of the chimeric DNA polymerases.
The hybrid genes for the chimeras were generated using the SOE (splicing by overlap extension) method as described in the Materials and methods section. In DNA sequencing, the chimera TaqEc1 showed the mutation A643G, which corresponds to the substitution of isoleucine 455 by valine in the thumb subdomain of the polymerase domain. As both residues are aliphatic and neutral, we suggest that these mutations have less or no influence on the polymerase activity. The chimeras TaqTne1 and TaqTne2 both contained one silent mutation in the modified region. In all cases large amounts of highly purified proteins were obtained. A total of 7.0 mg of protein with 98% purity of the enzyme TaqEc1 (Figure 2), a total of 4.5 mg of protein with 80% purity of the enzyme TaqTne1 and a total of 8.0 mg of protein with 90% purity of the enzyme TaqTne2 were isolated from 1 l of bacterial culture (Figure 3
). The enzyme fractions of the chimeras TaqEc1 and TaqTne2 showed the presence of double-stranded nucleases and DNA binding proteins.
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Neither the N-terminal nor the internal his tag seriously impairs the properties of the Taq DNA polymerase compared with the commercially available enzyme preparations (Lawyer et al., 1989). These results correspond to the observations described by Smirnov et al. (1995) for an N-terminal his-tagged Taq polymerase. The N-terminal his-tagged Taq DNA polymerase showed even slightly increased values of polymerase activity and temperature optimum (Table III
).
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The chimera TaqEc1 showed polymerase activity in the temperature range from 37 to 60°C with an optimum temperature of 50°C. The E.coli pol I has an optimum temperature around 37°C and the Taq polymerase around 72°C. Hence the temperature optimum of the chimera TaqEc1 is intermediate between the two parent polymerases. Korolev et al. (1995) compared the 3D structures of the Klenow fragment of the E.coli pol I and a truncated form of the Taq polymerase, called Klentaq1. They described several differences that may be part of the structural basis for the thermostability of Klentaq1. The comparison showed a large number of non-conserved amino acid substitutions, which indicates a global rearrangement of the charge distribution in Klentaq1. Furthermore, the contact area at the interface between the N-terminal and the large C-terminal polymerase domain is 230 Å2 larger in Klentaq1 and three additional ion pairs are formed at the interface. Additionally, their calculations showed that the surface is more hydrophobic in Klentaq1 and the electrostatic energy for the process of assembling the protein from individual amino acids in solution is significantly more favourable. For these reasons, it was not surprising that the polymerase domain of the Taq polymerase is destabilized in the chimera TaqEc1.
The chimera TaqTne1 showed polymerase activity in the temperature range from 50 to 60°C with an optimum temperature of 60°C. Thus, compared with the thermostable parental enzymes, thermostability is decreased (Tables III and IV). In comparison, the chimera TaqTne2, which contains the N-terminal
-helix of the polymerase domain of the Tne polymerase, showed an only slightly decreased temperature range from 50 to 80°C with an optimum temperature of 72°C. In conclusion, the specific polymerase activities of the chimeric DNA polymerases are decreased at the temperature optimum for each enzyme and increased around 5060°C compared with the Taq polymerase. Ignatov et al. (1997) reported that the replacement of 103 amino acids in the polymerase domain of the DNA polymerase from Thermus thermophilus by the respective fragment of E.coli pol I did not result in considerable changes of the specific activity. For this reason, we assume a connection between the increase in specific polymerase activity at 5060°C and the reconstruction of the 3'5' exonuclease domain in the Taq polymerase.
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The 3'5' exonuclease activity was assayed by degrading the 3'-terminus of a 5-end Dig-labeled primer annealed to a matrix strand. Of the three chimeric DNA polymerases, only the chimera TaqEc1 showed significant degradation of the matched and the mismatched primer (Figure 7). Compared with the N-terminal his-tagged Taq polymerase (Figure 6
), the chimera TaqEc1 clearly showed an inherent 3'5' exonuclease activity. Indeed, compared with the UlTma polymerase, which has 3'5' exonuclease activity (Figure 6
), the 3'5' exonuclease activity was weak and less specific. In order to test the coordination of the 3'5' exonuclease and the polymerase activity in the chimera TaqEc1, the 3'-mismatch primer correction assay was used. The digestion of DNA with EcoRI resulted in a 28 and an 18 bp fragment, even when the mismatched primers P2 and P3 were used (Figure 8
). Consequently, the mismatched nucleotides were replaced, which showed the inherent proof-reading function of the chimera TaqEc1. In comparison, the chimeras TaqTne1 and TaqTne2 showed no significant 3'5' exonuclease activity and no proof-reading function.
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However, one problem in exchanging large segments of polypeptides lies in the high probability that the network of interactions required for the structure and function of the protein will be perturbed. Nixon et al. (1998) and Béguin (1999) reviewed several examples that indicate that the folding is more likely to preserved when defined structural domains are exchanged compared with the shuffling of arbitrary polypeptide segments. In the Taq polymerase, the intervening and the polymerase domain stabilize each other. Consequently, it is not so easy to introduce a proof-reading function by exchanging the intervening domain without reducing the thermostability of the enzyme. Today, the structures of the Taq polymerase complexed with DNA and deoxyribonucleoside triphosphates (Eom et al., 1996; Li et al. 1998) are known, which give more insight into the cooperation of the 3'5' exonuclease and the polymerase activity. Probably a more sophisticated design based on a detailed knowledge of the structure and function or stochastic approaches will allow the introduction of a proof-reading function into the Taq polymerase while retaining high processivity, thermoactivity and thermostability.
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1 To whom correspondence should be addressed
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Received December 31, 1999; revised April 26, 2000; accepted May 17, 2000.