General reagents and equipment
Restriction enzymes and DNA ligase were purchased from New England Biolabs. Oligonucleotides were from Operon Technologies, Sigma-Genosys or Integrated DNA Technologies. DNA Engine Thermal Cyclers (MJ Research) were used for PCR. ÄKTAprime (Amersham Pharmacia Biotech) was used for protein purification.
Construction of Sso7d fusions
Construction of the Sso7d gene
. Overlapping oligonucleotides (Table ) with codons optimized for E.coli
expression were used to reconstruct the Sso7d
gene based on the published amino acid sequence (24
). Oligonucleotides Sso-W1, Sso-W2, Sso-C1, Sso-C2 and Sso-C3 were annealed at 10 µM each in the presence of 100 mM KOAc. The annealing mix was then added to a ligation mix to ligate Sso-C1, Sso-C2 and Sso-C3 with the aid of the bridging oligonucleotides Sso-W1 and Sso-W2. Oligonucleotides Sso-R1 and Sso-Xba were used to amplify the full-length ligated product in a PCR. The amplified DNA encodes the Sso7d
gene juxtaposed by the appropriate restriction sites for the subsequent steps.
S-Taq(Δ289) fusion. Plasmid pUTAQ was generated by inserting the DNA fragment containing the T.aquaticus PolI gene into vector pUC18 at the polylinker site. The PCR fragment containing the synthetic gene for Sso7d was cloned into pUTAQ to replace the region encoding the first 289 amino acids of Taq polymerase. The resulting construct, pYW1, overexpresses the S-Taq(Δ289) gene from lacI promoter upon induction with IPTG.
S-Taq fusion. A PCR fragment encoding the first 289 amino acids of Taq polymerase was amplified and inserted back into plasmid pYW1 (see above) at the junction between Sso7d and Taq(Δ289). The resulting plasmid, pYW2, allows the expression of a polypeptide (S-Taq) containing the Sso7d protein fused to the N-terminus of full-length Taq.
Mutant Sso7d fusions. An oligonucleotide containing a degenerate codon (GNG) at the position corresponding to Trp24 in Sso7d (Fig. A) was used to introduce point mutations via two-step sequential PCR. The final PCR fragment encoding the Sso7d gene with the degenerate codon at position 24 was inserted into pYW1 to replace the wild-type Sso7d gene. The resulting plasmids encode one of the following four amino acids, Gly (GGG), Val (GTG), Glu (GAG) or Ala (GCG), at the position corresponding to Trp24 in the wild-type Sso7d protein.
(A) Amino acid sequence of Sso7d protein. (B) Schematic representation of the domain organization of Taq polymerase and the Sso7d fusion proteins.
All Taq-based Sso7d fusion proteins contained a 6-His affinity tag at the C-terminus to facilitate protein purification.
Pfu-S fusion. A plasmid (pETPFU) carrying the Pfu DNA polymerase gene under the control of the T7 promoter was modified so that unique restriction sites were introduced at the 3′ end of the Pfu gene. The resulting plasmid (pPFKS) expresses a Pfu polymerase (Pfks) with three additional amino acids (Gly-Thr-His) at its C-terminus. No functional difference was observed between Pfks and commercial Pfu polymerase (Stratagene). The Sso7d gene was PCR amplified and inserted into the pPFKS plasmid. The resulting plasmid, pPFS, overexpresses a single polypeptide (Pfu-S) containing Sso7d protein fused to the C-terminus of Pfu polymerase. The identity of the overexpressed protein was determined by both the apparent molecular weight on SDS–PAGE and its cross-reactivity with anti-Sso7d antibodies.
Expression and purification of Sso7d fusion proteins
Plasmid DNAs encoding the Taq-based Sso7d fusion proteins were transformed into E.coli
strain BL21 (Stratagene) and grown in 500 ml of 2× YT medium in the presence of carbenicillin to an OD600
of 0.3. IPTG was added to 1 mM to induce expression from the lac
promoter. Cells were harvested 3–4 h later. Cleared lysate was prepared as reported previously (25
). Solid ammonium sulfate was added to 70% (w/v, 4°C) to precipitate the majority of the soluble proteins in the lysate.
The ammonium sulfate pellet was dissolved in Ni–NTA binding buffer NiB (20 mM Tris–HCl pH 7.9, 5 mM imidazole pH 7.5, 5 mM 2-mercaptoethanol, 0.1% NP40 and 500 mM KCl) and loaded onto a pre-equilibrated Ni–NTA (Qiagen) column (1 ml). The bound protein was step eluted using buffer IM-150 (NiB buffer with 150 mM imidazole). The fractions were pooled and purified on a heparin–agarose column by binding in buffer HP-100 (20 mM Tris-HCl pH 7.9, 0.1% NP40 and 100 mM KCl) and eluting with buffer HP-500 (HP-100 with 500 mM NaCl). The peak fractions were pooled and concentrated via a second Ni–NTA column before dialyzing (Slidelyzer; Pierce) against a pre-storage buffer (50 mM Tris–HCl pH 7.9, 250 mM KCl, 0.25 mM EDTA, 2.5 mM DTT, 0.1% NP40 and 0.1% Tween 20). Glycerol, NP40 and Tween 20 were added to the dialyzed sample so that the final storage buffer contained 20 mM Tris–HCl (pH 7.9), 100 mM KCl, 0.1 mM EDTA, 1 mM DTT, 0.5% NP40, 0.5% Tween 20 and 50% glycerol.
Expression of Pfu-S was performed similarly as described above using strain BL21(LysS). Both carbenicillin and chloroamphenicol were added to the growth media. The ammonium sulfate precipitate was resuspended and dialyzed against buffer Q-50 (20 mM Tris–HCl pH 7.0, 50 mM NaCl and 5 mM 2-mercaptoethanol) before loading onto a 1 ml HiTrapQ column (Amersham Pharmacia Biotech). The flow-through was collected and purified on a 1 ml P11 phosphocellulose column (Whatman). The bound protein was eluted with Q-350 buffer (Q-50 buffer with 350 mM NaCl). The peak fractions were dialyzed against Q-50 buffer before loading onto a 1 ml HiTrapSP column (Amersham Pharmacia Biotech). The polymerase was eluted with a 50–800 mM NaCl gradient. The peak fractions, which corresponded to elution with 240–320 mM NaCl, were pooled and dialyzed against a pre-storage buffer (125 mM Tris–HCl pH 8.2, 0.25 mM EDTA, 2.5 mM DTT, 0.1% NP40, and 0.1% Tween 20). Glycerol, NP40 and Tween 20 were added to the dialyzed sample so that the final storage buffer contained 50 mM Tris–HCl pH 8.2, 0.1 mM EDTA, 1 mM DTT, 0.5% NP40, 0.5% Tween 20 and 50% glycerol.
Polymerase activity assay
An oligonucleotide (–47M13L, Table ) at 0.8 µM was pre-annealed to ssM13mp18 DNA (40 nM) (Bayou Biolab) in a 2.5× reaction buffer (25 mM Tris–HCl pH 8.8, 125 mM KCl and 0.25% Triton X-100) before mixing with dNTPs and DNA polymerase. MgCl2
was added to initiate DNA synthesis at 72°C. The final reaction contained 10 mM Tris–HCl (pH 8.8), 50 mM KCl, 2 mM MgCl2
, 200 µM dNTPs, 0.1% Triton X-100 and 16 nM primed template. Samples were taken at various time points and added to a 1:200 dilution of PicoGreen (Molecular Probes) in TE (10 mM Tris–HCl pH 8.0 and 1.0 mM EDTA pH 8.0). The amount of DNA synthesized was quantified using a fluorescence plate reader (BioTek Instruments). The unit activity of the DNA polymerase of interest was determined by comparing its initial rate with that of a control DNA polymerase (e.g. AmpliTaq, from Applied Biosystems). For S-Taq(Δ289), Taq, S-Taq, Pfu-S and the mutant Sso7d fusion proteins, the unit activity determined by this method agreed well (within 2-fold) with that determined by the conventional radioactive unit definition assay (26
; T. Tenkanen and T. Soininen, personal communication). When significant differences were observed for Taq(Δ289) and Pfu between the two assays, units were defined using the conventional method.
A 5′ FAM-labeled primer (50 nM) (–40M13LFF, Table ) was added to ssM13mp18 DNA (100 nM) in the presence of 10 mM Tris–HCl pH 8.8, 50 mM KCl, 2.5 mM MgCl2
, 250 µM dNTPs and 0.1% Triton X-100, unless otherwise indicated. Primer annealing to the DNA template was achieved by heating at 90°C for 5 min, cooling to 72°C at 0.1°C/s, incubating at 72°C for 10 min, cooling again to 4°C at 0.1°C/s, using a DNA Engine thermal cycler (MJ Research). Then 4 μl of DNA polymerase (diluted in 10 mM Tris–HCl pH 8.8, 50 mM KCl and 0.1% Triton X-100) was then added to 16 μl of primed template at a molar ratio of 1:500–1:10 000 to initiate DNA synthesis at 72°C. Samples were diluted in gel loading dye and analyzed on a MJ BaseStation Sequencer (MJ Research). To ensure that no multiple binding/extension occurs on any primer–template complexes, both the polymerase concentration and the reaction time were varied, and the median product length was determined for each reaction. The median product length is defined as the length of the product at which the total fluorescence intensity of all products up to that length equals 50% of the sum fluorescence intensity of all detectable products. In general, a higher ratio of primer–template to polymerase concentration is necessary to achieve processive conditions for low processivity enzymes, whereas for high processivity enzymes a lower ratio is needed to detect longer primer extension products under processive conditions (see corresponding figure legends). When the median product length no longer changes with an increase in reaction time or a decrease in polymerase concentration, the traces of those samples were used to determine the processivity using the analysis method described by von Hippel et al
). Each peak with a signal level significantly above the background was integrated to obtain the intensity at each position (nI
) and the total peak intensity (nT
) of all detectable products. The integration data were plotted as log(nI
) versus n
– 1, where n
is the number of nucleotide residues incorporated, and fitted to the following equation:
log(nI/nT) = (n – 1)logPI + log(1 – PI)
where PI represents the probability of not terminating at position I and is defined as the ‘microscopic processivity parameter’ for this position. The average primer extension length was determined from 1/(1 – PI).
Steady-state kinetic analyses
Steady-state kinetic analyses were performed using the polymerase activity assay described above with the following modifications. Each reaction contained 1.2 nM enzyme. The molar concentration of the enzyme was determined using a combination of the Bradford assay (Bio-Rad) and SDS–PAGE analysis. The pre-annealed primed ssM13 template was used at 1–12 nM for S-Taq(Δ289), Taq and S-Taq, 5–30 nM for Taq(Δ289 and 0.2–6 nM for Pfu and Pfu-S. Buffers with two different salt concentrations were used. For Taq(Δ289), S-Taq(Δ289), Pfu and Pfu-S the buffer contained 10 mM Tris–HCl (pH 8.8), 10 mM KCl, 2 mM MgCl2 and 0.1% Triton X-100. For Taq and S-Taq the buffer contained 50 mM KCl and was otherwise identical to the first buffer. Then, dNTPs were added at 200 µM each. The initial rate of each reaction was plotted against primer–template concentration and fitted to the following equation:
V = kcat × [E] × [D]/(Km (DNA) + [D])
where V is the initial rate, [D] is the primer–template concentration, [E] is the enzyme concentration, kcat is the turnover rate and Km (DNA) is the primer–template concentration at which half of the maximum activity is achieved.
Thermal stability assay
Each enzyme (aliquoted in 30 µl at 40 U/ml) was first incubated at 97.5°C for varied periods of time in the presence of the corresponding optimal reaction buffer as indicated in the figure legends. At each time point, the heated sample was transferred to a 72°C thermal block and allowed to equilibrate for 1 min. Then, 20 µl of the sample was mixed with an equal volume of 2× polymerase activity assay reaction mix pre-heated to 72°C (see above). The remaining activity was plotted versus time spent at 97.5°C. The data were fitted to the following equation:
ln(A) = ln(A0) – kt
where A represents the remaining activity at time t, A0 is the activity at t = 0 and k is the rate constant for enzyme inactivation. The half-life (t1/2) was determined from the following equation:
t1/2 = (ln2)/k
PCR efficiency assay
λ DNA (130 pg/µl) was used as the template to assess the relative efficiency of each polymerase in a PCR. For extension efficiency comparison, a set of primers (see Table ) was used to amplify amplicons of 0.5, 1, 2, 5, 8, 10, 12 and 15 kb in size from the template in a 20 µl reaction. For the salt dependence comparison experiment, a single pair of primers (see Table ) that amplifies a 0.9 kb amplicon was used for all reactions. The amount of enzyme, the reaction buffer for each enzyme and the cycling protocol used are indicated in the corresponding figure legends. It was necessary to use a higher amount of the low efficiency polymerases [e.g. Taq(Δ289) and Pfu] so that a sufficient amount of DNA could be generated to allow comparison. Upon completion of the PCR, 5 µl of the PCR was mixed with loading dye and loaded onto a 1% agarose gel. The gel was stained with ethidium bromide. A Kodak gel documentation system was used to photograph the gel.