A fusion of Taq DNA polymerase with the CL7 protein from Escherichia coli remarkably improves DNA amplication

DNA polymerases are important enzymes that synthesize DNA molecules and therefore are critical to various scientic elds as essential components of in vitro DNA synthesis reactions, including PCR. Modern diagnostics, molecular biology, and genetic engineering require DNA polymerases with improved performance. This study aimed to obtain and characterize a new CL7-Taq fusion DNA polymerase, in which the DNA coding sequence of Taq DNA polymerase was fused with that of CL7, a double-stranded DNA binding-like protein from Escherichia coli. The resulting novel recombinant gene was cloned and expressed in E. coli. The recombinant CL7-Taq protein exhibited excellent thermostability, extension rate, sensitivity, and resistance to PCR inhibitors. Our results showed that the sensitivity of CL7-Taq DNA polymerase was 100-fold higher than that of wild-type Taq, which required a template concentration of at least 1.8 × 10 5 aM. Moreover, the extension rate of CL7-Taq was 4 kb/min, which remarkably exceeded the rate of Taq DNA polymerase (2 kb/min). Furthermore, the CL7 fusion protein showed increased resistance to inhibitors of DNA amplication, including lactoferrin, heparin, and blood. Single-cope human genomic targets were readily available from whole blood, and pretreatment to purify the template DNA was not required.

than 1 kb due to the relatively low processivity and thermostability of the wild-type enzyme. Generally, the e ciency of ampli cation by Taq polymerase for targets shorter than 1 kb is approximately 80% [4]. However, Taq DNA polymerases become completely inhibited when the PCR mixture contains 0.2% blood. It seems that hemoglobin and lactoferrin have important roles in inhibiting the ampli cation process [5].
Therefore, these drawbacks limit the applications of Taq DNA polymerase.
Recently, a novel strategy was employed to overcome these limitations [6]. Generating fusion proteins with Taq DNA polymerases and a thermostable DNA-binding protein such as the Sso7d DNA-binding protein from Sulfolobus solfataricus has been shown to enhance the processivity [7], thermostability, and overall stability compared with wild type Taq polymerase. The puri ed S-Taq protein has shown acceptable limits of host genomic DNA levels without the use of DNases or other DNA precipitating agents, which highlights its potential for use in PCR-based diagnostics, in-situ PCR, and forensic science [7].
A previous study also found that adding 0.6% bovine serum albumin (BSA) to reaction mixtures containing Taq DNA polymerase reduced the inhibitory effect of blood and allowed DNA ampli cation in the presence of 2% instead of 0.2% blood [8]. Furthermore, BSA was found to be the most e cient ampli cation facilitator. As rapid and simple diagnostics methods are urgently required for blood analyses, modi cations to Taq DNA polymerase that enhance its tolerance to inhibition by blood are signi cant to medical diagnostics. Mutations to Taq DNA polymerase that render it resistant to inhibition by blood components are now commercially available [9]. Additionally, there are Taq polymerase mutants that are used in PCR-based tests of blood and soil samples that are widely used for diagnostics and forensic analyses and do not require pretreatment to purify the template DNA and others that allow increased dye concentration overcomes uorescence background and quenching in real-time PCR analyses of blood. Because of de ciencies in the 3 -5 exonuclease domain, Taq DNA polymerase is widely applied to ARMS-PCR to detect single nucleotide polymorphisms (SNPs).
In recently reported studies, Taq polymerase was fused to DNA binding proteins to improve other properties [10]. To further improve the properties of Taq polymerase, we fused the DNA binding protein of CL7, which is a mutant of the CE7 protein derived from E. coli, to the Taq DNA polymerase with a 7-amino acid linker [11]. We demonstrated and functionally characterized the fusion enzymes and demonstrated that they showed improved processivity, sensitivity, ampli cation rates, and eliminated pre-PCR treatment steps. Finally, we demonstrated the practical bene ts for PCR applications of enhancing the sensitivity of the polymerase.

Results
Expression and puri cation of Taq and CL7-Taq To obtain the puri ed enzymes, we designed the experiment as follows. The gene encoding a fragment of Taq DNA polymerase was cloned into the pET-30 vector to generate a pET30/Taq plasmid. This led to the expression of a Taq DNA polymerase fusion protein with a C-terminal 6 × histidine tag. To achieve fusion with the CL7 gene, the two PCR products were mixed together with the pET-30 vector to generate the pET30/CL7-Taq plasmid, which encoded the fusion enzyme with a C-terminal 6 × histidine tag. Then the pET30/Taq and pET30/CL7-Taq plasmids were transformed into E. coli BL21 (DE3) to express the Cterminally His-tagged Taq and CL7-Taq proteins, respectively. Following expression, the cells were harvested and sonicated. The recombinant DNA polymerases were then puri ed by passing the heatdenatured supernatant through a His-Bind Ni 2+ a nity column. After each puri cation step, the purity of the DNA polymerase was monitored by SDS-PAGE (Fig. 1), which separated the following major protein bands: 93 and 108 kDa for Taq and CL7-Taq, respectively; this result was in agreement with the molecular masses of 92.7 and 107.7 kDa that were calculated based on the amino acid sequences. The E.coli overexpression system used in this study enabled the production of 720 mg of Taq polymerase and 620 mg of CL7-Taq fusion protein per 1 L of induced culture. After being measured by the EvaEZ Fluorometric Polymerase Activity Assay Kit, the speci c activities of the puri ed Taq and CL7-Taq polymerases were found to be 1426.4 and 1572.6 U/mg, respectively. These results indicated that the CL7 fusion had a positive effect on the relative activity of Taq DNA polymerase. The production e ciency of Taq and CL7-Taq DNA polymerase in this study were satisfactory.

Characterization of Taq and CL7-Taq
In all experiments, the activity of 1 µL of enzyme was determined for Taq and CL7-Taq DNA polymerases and compared with commercial Taq polymerase with an activity of 1 U/µL using the EvaEZ Fluorometric Polymerase Activity Assay Kit (Biotium) in an isothermal reaction at 60 °C on a real-time PCR apparatus (Bio-Rad). For characterization purposes, the activity of Taq and CL7-Taq DNA polymerases were measured by PCR using various buffer compositions and concentrations of MgCl 2 , KCl, and (NH 4 ) 2 SO 4 and various pHs (Fig. 2).
The effect of pH on the activity of Taq and CL7-Taq DNA polymerases was evaluated using Tris-HCl buffers of pH ranging from 7.0 to 9.0. Both polymerases had the highest enzyme activity at pH 8.0 (Fig. 2c). The activity of Taq and CL7-Taq DNA polymerases were closely related to the concentration of MgCl 2 , which was optimal at between 1 to 8 mM and 2 to 8 mM, respectively (Fig. 2a). DNA polymerase activity was completely inhibited when KCl concentrations surpassed 70 mM for Taq and 80 mM for CL7-Taq (Fig. 2b). The activity of Taq and CL7-Taq DNA polymerase were also strongly affected by (NH 4 ) 2 SO 4 and were completely inhibited at concentrations over 30 and 40 mM, respectively (Fig. 2d). The fusion of CL7 to Taq DNA polymerase resulted in higher tolerance of the enzyme to salt inhibition.

PCR ampli cation rate and processivity
To measure the PCR ampli cation rate, we designed tests similar to those previously published [15]. The results showed that the fusion CL7-Taq DNA polymerase replicated template strands at a faster rate than the Taq DNA polymerase (Fig. 3). The data showed thatCL7-Taq extended a 4,000-bp product during 60 s, while Taq DNA polymerase required 1 min for a 2,000-bp product. This showed that the fusion of CL7 protein with Taq DNA polymerase was twofold more e cient than Taq DNA polymerase without CL7, meaning DNA ampli cation required less time.

Sensitivity
To monitor enzyme sensitivity, we used the protocol published before [15]. Sensitivity was measured by PCR with CL7-Taq and Taq polymerases and 10-fold serial dilutions of template; the product size was 3,000 bp. The results showed that the fusion polymerase was more sensitive than the wide-type polymerase. In the case of CL7-Taq polymerase, it was su cient to use 1.8 × 10 3 aM of plasmid DNA, while Taq polymerase required at least 1.8 × 10 5 aM of plasmid. These data showed that the sensitivity of CL7-Taq DNA polymerase was improved 100-fold compared with Taq polymerase (Fig. 4).

Thermostability of the DNA polymerases
To determine the thermostability of Taq and CL7-Taq DNA polymerases, the enzymes were monitored for decreased activity after preincubation at 95 °C and 99 °C. These experiments revealed that the CL7-Taq DNA polymerase had remarkably higher thermostability. Our data showed that Taq and CL7-Taq DNA polymerase were functional after 30 and 50 min at 99 °C, respectively (Fig. 5a). Additionally, Taq and CL7-Taq DNA polymerases remained active after 2 and 3 h at 95 °C, respectively (Fig. 5b).

Tolerance of the DNA polymerases to PCR inhibitors
To determine the limiting concentration of PCR inhibitors, the fusion CL7-Taq and the Taq DNA polymerases were PCR-tested for their resistance to serial dilutions of blood, lactoferrin, and heparin, which have been reported to be PCR inhibitors in many publications (Kermekchiev et al. 2008). The results showed that the CL7-Taq and Taq polymerase remained active in the presence of 3.5 µg and 2 µg lactoferrin, respectively (Fig. 6b), and 14 µg and 6 µg of heparin, respectively (Fig. 6a). CL7-Taq and Taq were resistant to 2% and 0% blood, respectively (Fig. 6c). Together, these ndings revealed that the fusion CL7-Taq DNA polymerase was signi cantly more resistant to blood, lactoferrin, and heparin compared with the Taq enzyme.
Discussion PCR technology has been widely applied in the elds of molecular biology, genetic engineering, and diagnostics [8]. PCR ampli cation e ciency is strongly dependent on the properties of the DNA polymerase and reaction conditions. Wild-type DNA polymerase has some drawbacks; therefore, modern diagnostic methods and genetic engineering techniques require modi ed DNA polymerases with better properties, such as those that possess higher sensitivity and/or ampli cation rates. For this reason, we engineered fusions of DNA polymerases with single and double stranded DNA binding proteins to enhance the DNA binding ability.
In this study, the N-terminus of Taq DNA polymerase was fused with the thermoduric CL7 protein using a 7-amino acid linker (Gly-Asn-Let-Tyr-Phe-Gln-Cys). It has been reported that the template is picked up more selectively from the mixture in the presence of a binding domain and that PCR is more e cient even when inhibitors are present [4] Some studies have shown that covalently linking a DNA binding protein to a DNA polymerase can strongly enhance polymerase activity [3,6]. Similar results are shown in this study, in which the CL7 protein doubled the elongation rate and increased sensitivity for the DNA template by 100-fold.
The most common challenges during ampli cation of environmental and blood samples are inhibitors present in the tested material [17]. Our observations of successful ampli cation of the β-actin gene directly from human blood using CL7-Taq is very promising and opens up a new avenue for this polymerase as a valuable tool for medical diagnostics and forensic sciences, where sample availability is minimal. We found that the enhanced salt tolerance of CL7-Taq is responsible for its successful use in direct PCR of the human genome without preprocessing. Previous studies have shown that PCR inhibitors alter DNA or block enzymes though these pathways, such as inhibiting the active site or blocking access to the active site for cofactors such as Mg 2+ ions [8,18]. Therefore inhibitors either weaken the e ciency of PCR ampli cation or block it completely. However, commercially available native enzymes are not always able to deal with these PCR issues. Our data showed that the fusion CL7-Taq DNA polymerase exhibited a higher tolerance to PCR inhibitors (blood, lactoferrin, and heparin) compared with Taq DNA polymerase. Therefore Taq DNA polymerase containing CL7 had better performance in the ampli cation of complicated templates.
Our data showed that the fusion of CL7 to Taq DNA polymerase improved enzymatic properties such as thermostability, ampli cation rate, and template sensitivity. In thermostable tests, we found that the thermostability of CL7-Taq DNA polymerase was remarkably higher than Taq DNA polymerase. The thermostability of CL7-Taq was over 1 h longer than that of Taq DNA polymerase at 95 °C. Mg 2+ and other salts were critical components of PCR reactions; thus, optimizing their concentrations was essential for native DNA polymerase. Unlike Taq DNA polymerase, CL7-Taq DNA polymerase exhibited su cient ampli cation e ciency within a wide range of Mg 2+ concentrations. Furthermore, CL7-Taq DNA polymerase had a satisfactory ampli cation e ciency at various concentrations of KCl and (NH 4 ) 2 SO 4 .
Thus, the salt tolerance of CL7-Taq was remarkably enhanced.
Our observation that CL7-Taq had higher sensitivity than Taq makes it an attractive enzyme for PCRbased diagnostics. Therefore, it is tempting to speculate that one could explore using CL7-Taq for ARMS-PCR to detect SNPs or common DNA viruses such as HPV, Herpesvirus, and Parvoviruses.

Conclusions
In this study, we aimed to obtain and characterize a new CL7-Taq fusion DNA polymerase, in which the DNA coding sequence of Taq DNA polymerase was fused with that of CL7, a double-stranded DNA binding-like protein from Escherichia coli. Our results showed that the sensitivity of CL7-Taq DNA polymerase was 100-fold higher than that of wild-type Taq, which required a template concentration of at least 1.8 × 105 aM. Moreover, the extension rate of CL7-Taq was 4 kb/min, which remarkably exceeded the rate of Taq DNA polymerase (2 kb/min). The recombinant CL7-Taq protein exhibited excellent thermostability, extension rate, sensitivity, and resistance to PCR inhibitors. Thus, this is a novel enzyme that improved the properties of Taq DNA polymerase, and thus may have wide application in molecular biology and diagnostics.

Construction of recombinant plasmids
First, DNA coding sequences of the DNA polymerase from Thermus aquaticus (GenBank: P19821.1) and of CL7, which is a mutant of CE7 from E. coli (GenBank: CP018986.1) were obtained [11]. The two fragments were synthesized by GeneCreate (Wuhan, China). The designed primers for ampli cation are listed in Table 1. The gene synthesis procedure was performed as described previously. PCR ampli cation was then used to obtain two products: the Taq DNA polymerase gene (2493 bp) and the CL7 gene (390 bp). The pET-30 vector was digested with the restriction endonuclease Xba I (Takara, Shiga, Japan), and the product was puri ed using the DNA Gel Extraction Kit (Promega, Madison, WI, USA). Then the PCR products were mixed together with the digested pET-30 vector. Finally, T5 cloning (New England Biolabs, Ipswich, MA, USA) was performed, in which CL7 was fused to the N-terminus of Taq DNA polymerase with the 7-amino acid linker (ENLYFQG) and a 6 × His tag to the C-terminus. This was necessary for the puri cation of recombinant protein by metal a nity chromatography. Nucleotide sequences of the resulting recombinant plasmids, pET30/Taq and pET30/CL7-Taq, were con rmed by DNA sequencing (Sangon, Shanghai, China). containing 50 µg/mL kanamycin at 37 °C. Then isopropyl-β-D-1-thiogalactopyranoside (IPTG) was added at a nal concentration of 1 mg/ml to induce Taq or CL7-Taq expression from the T7 promoter with shaking at 18 °C. After 12 h incubation, the cells were centrifuged at 7,000 × g for 5 min, and the pellets were resuspended in 20 mL of lysis buffer (20 mM Tris-HCl [pH 9.0], 0.5 M NaCl, and 10 mM imidazole). Protein complexes were extracted by ultrasonic decomposition and the insoluble debris was removed by centrifugation at 12,000 × g and 4 °C for 20 min.
For heat treatment, the cleared lysate was immersed in a 75 °C orbital water shaker for 30 min, cooled on ice for 20 min, and then the denatured host proteins were removed by centrifugation at 12,000 × g and 4 °C for 20 min. Following heat treatment, the exogenous proteins were puri ed in a one-step process DNA polymerase activity assay The DNA polymerase activity of puri ed proteins was assayed using the EvaEZ Fluorometric Polymerase Activity Assay Kit (Biotium, Hayward, CA, USA). All assays were conducted in an isothermal reaction at 60 °C using a CFX Real-Time PCR instrument (Bio-Rad, Hercules, CA, USA) in accordance with the de nition of one unit of enzyme activity ("One unit of DNA polymerase activity is usually de ned as the amount of enzyme that will produce 10 nmol of nucleotides during a 30-min incubation"). Enzymatic activity was determined relative to a commercial Taq DNA polymerase (Thermo Fisher Scienti c, Waltham, MA, USA) with an activity of 1 U/µL. When the DNA polymerase was active, the primer was extended to form a double-stranded product that bound the EvaGreen dye, resulting in increased uorescence. The rate of increase is positively correlated with polymerase activity [13].

Optimization of PCR ampli cation
To optimize the ampli cation process, polymerase activity was measured using various concentrations of MgCl 2 , KCl, and (NH 4 ) 2 SO 4 in the buffer as well as various pHs. All PCR reactions were performed Temperature stability was also assayed [14]. One unit of puri ed CL7-Taq and Taq DNA polymerases were heated at 99 °C for 10, 20, 30, 40, 50, and 60 min, and at 95 °C for 1, 2, 3, 4, and 5 h. Then, the same amount of enzyme was used to amplify a 445-bp target fragment under the optimal reaction buffer (in the same PCR conditions determined in the optimization process): 20 mM Tris-HCl (pH 8.0), 3 mM MgCl 2 , 10 mM (NH 4 ) 2 SO 4 , and 20 mM KCl.
Measuring the PCR ampli cation rate PCR ampli cation rates were measured using the protocol described by [15] CL7-Taq and Taq DNA polymerases were used to amplify PCR products of 1, 2, 3, and 4 kb under the conditions determined in the optimization process and using pET23a/dcas9 plasmid DNA as a template, which was recombined in our laboratory. PCR ampli cation started with an initial denaturation at 95 °C for 3 min and included 25 cycles of 30 s at 95 °C, 30 s at 60 °C, and 60 s at 72 °C.

Sensitivity
Following su cient modi cations, DNA polymerase sensitivity (a nity for template) was measured using the protocol by [16]. PCR was conducted under conditions optimized for the fusion polymerases CL7-Taq and Taq. We used pET23a-GFP Plasmid as a template along with the primers 5 -TGGTCTTCAATGCTTTGCGAGATAA-3 (forward) and 5 -CTTTTCGTTGGGATCTTTCG-3 (reverse). The product of the reaction was 445 bp. The reaction was conducted at decreasing template concentrations (serial 10-fold dilutions of the template) and included an initial denaturation at 95 °C for 3 min followed by 35 cycles of 30 s at 95 °C, 30 s at 60 °C, and 90 s at 72 °C. The ampli ed fragments were analyzed in a 1.5% agarose gel stained with ethidium bromide.

Resistance to inhibitors
The effect of PCR inhibitors such as heparin (Sigma-Aldrich, St. Louis, MO, USA) at a range from 16 to 1 µg, lactoferrin (Sigma-Aldrich) at a range from 4 to 0.5 µg, and blood at a range from 8-1%, on the catalytic activity of the CL7-Taq and Taq DNA polymerases was assessed by a PCR reaction using human genomic DNA as a template and the speci c β-actin primers 5 -AGAGATGGCCACGGCTGCTT-3 (forward) and 5 -ATTTGCGGTGGACGATGGAG-3 (reverse) [16]. The ampli ed fragments were analyzed in a 1.5% agarose gel stained with ethidium bromide.   Comparison of the PCR ampli cation rates of the fusion Cl7-Taq DNA polymerase (b) and Taq DNA polymerase (a). The elongation times used for PCR ampli cation are shown at the top. LaneM is the GeneRuler 1 kb DNA ladder marker (250-1,0000 bp) (Thermo Fisher Scienti c). The ampli ed products were analyzed in a 0.8% agarose gel stained with ethidium bromide.    The effect of heparin (a), lactoferrin (b), and blood (c) on DNA ampli cation using human genomic DNA as a template and primers for β-actin. Control reactions were performed without any inhibitors. Lane M, DL2000 (100-2000 bp) (TsingKe, China). The ampli ed products were analyzed in a 1 % agarose gel stained with ethidium bromide.

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