Cloning and Expression of Thermus aquaticus DNA polymerase in Escherichia coli

Headnote

Abstract

Thermostable DNA polymerase gene from Thermus aquaticus was cloned into constructed Taq from Thermus a Qaticus (pTTQ) plasmid using EcoRI and SalI sites with subsequent transformation in Escherichia coli strain (TOP10). The use of Isopropyl-β-D-thiogalactopyranosid (IPTG) as inducer of interested gene expression under control of the lac promoter was investigated. The optimization of enzyme induction by IPTG was determined at shake flask level to be 0.52mM at exponential growth phase. Enzyme preparation was performed by lysis the cultured cells. Afterwards, the cell suspension was incubated at 75°C to denature all heat sensitive proteins in the cell suspension that have been removed by subsequent centrifugation. Finally, the clarified supernatant containing heat resistant Taq DNA polymerase was collected and stored at -80°C. The activity of enzyme was compared with commercial Taq DNA polymerase, which remained when stored in buffer containing 50% glycerol, at -20°C. The purified enzyme had a molecular weight of 94 KDa, as estimated by SDS-PAGE and yielded appropriate enzyme activity comparing to the commercial Taq DNA polymerase.

Keywords: Taq DNA Polymerase, E. coli, expression, Thermus aquaticus

Introduction

Thermostable DNA polymerase is a very important enzyme for molecular biological studies such as DNA amplification and DNA sequencing by the polymerase chain reaction (PCR) [1,2]. Most of the thermostable DNA polymerases have been isolated from Thermus aquaticus, a thermostable bacterium, known as Taq polymerase. Taq DNA polymerase is an enzyme obtained from a heat stable bacterium called T. aquaticus having a molecular weight of about 6.6?104-9.4?104 Daltons [3]. T aquaticus is a bacterium that lives in hot springs and hydrothermal vents [4]. Taq polymerase was identified as an enzyme able to withstand the protein-denaturing conditions (high temperature) required during PCR [5]. Therefore it replaced the DNA polymerase from E. coli originally used in PCR [6]. Taq's temperature optimum for activity is 75-80°C, with a half-life of 9 minutes at 97.5°C, and can replicate a 103 base pair strand of DNA in less than 10 seconds at 72°C [7].

Taq DNA polymerase catalyzes the incorporation of dNTPs into DNA. It requires a DNA template, a primer terminus, and the divalent cation Mg++. Taq polymerase contains a polymerization dependent 5'-3' exonuclease activity. It does not have a 3'-5' exonuclease and thus no proof reading function. Despite this, the enzyme synthesizes DNA invitro with reasonable fidelity [8]. Use of the thermostable Taq polymerase eliminates the need for having to add new enzyme to the PCR reaction during the thermocycling process. A single closed tube in a relatively simple machine can be used to carry out the entire process. Thus, the use of Taq polymerase was the key idea that made PCR applicable to a large variety of molecular biology problems concerning DNA analysis [5].

The Taq DNA polymerase isolated from T. aquaticus was the first characterized thermostable enzyme but more than 50 DNA polymerase genes have been cloned and sequenced from various organisms including thermophiles by PCR cloning technique, whereby the gene encoding this enzyme was cloned into the expression vectors that produce recombinant Taq polymerase gene has facilitated for this enzyme production [3]. The recombinant Taq DNA polymerase expressed in E. coli shows identical characteristics to native Taq from T. aquaticus with respect to activity, specificity, thermostability and performance in PCR [9]. However, the lac promoter and its derivatives are widely employed for the purposes mentioned above, and in most cases, IPTG is used as inducer for foreign gene expression [4, 5]. Our goal, in the present study, is the cloning and expression of recombinant Taq DNA polymerase in the E. coli for performance in PCR.

Materials and Methods

Molecular cloning of the gene for Taq DNA polymerase

Genomic DNA of T. aquaticus and plasmid DNA were isolated by a method adapted from Sambrook [7]. A 2.6 Kb fragment containing the whole T. aquaticus DNA polymerase gene was prepared by PCR amplification [2] with the T. aquaticus genomic DNA using primers forward-primer 5' - CGG AAT TCT GAG GAG GTA ACA TGA GGG-3' and the reverse-primer sequence 5'-CGT CGA CTA GAT CAC TCC TTG GCG GAG AG -3' which created the underlined unique EcoRl and Sail restriction sites respectively at each end of the amplified DNA fragment. The primer sequences were adopted as described [9]. The fragment was ligated into the expression vector pET (invitrogene) that had been digested before with EcoRl and Sail (Sigma), giving a closed circular fusion molecule (The constracted vector have been called pTTQ. The ligate was transformed into competent E. coli strain, TOP 10 (Sinagen) by CaCb (Sigma) using heat shock method at 42°C for 45 seconds [8].

Culture and expression conditions

The recombinant E. coli was cultured in 10 ml of Lauria Bertaini (LB) broth (Merck) overnight at 3 7° C containing 100µg/ml ampicillin (Merck) as seed culture. The LB medium containing 100µg/ml ampicillin was inoculated with 1% of seed culture (250ml of LB for shake flask system) and grown at 37°C. The expression of recombinant protein was induced by 0.52mM IPTG (Sigma) to the growing culture at an OD^sub 600^ of 0.6-0.8. The culture was continued overnight. No addition of inducer was used as negative control experiment [10].

Enzyme extraction and purification

The cells were harvested by centrifugation at high speed, washed, and then resuspended in buffer A [(I mM EDTA; Sigma), 50 mM Tris-HCl (pH: 8.0) (Sigma), 50 mM Glucose (Merck)] and buffer B [1 mM EDTA, 50 mM Tris-HCl (pH: 8.0), 50 mM Glucose, 4 mg/ml Lysozyme, Sigma] to a twentieth of the culture volume. The cells were then lysed by adding 15ml of lysis buffer [10 mM Tris-HCl (pH: 8.0), 0.1 mM EDTA, 0.5% Tween 20, Merck, 0.5% Nonidet P40, Sigma, 50 mM KCl, Sigma, 1.0 mM PMSF, Sigma]. The suspension was incubated for Ih at 75°C. Cellular debris was removed by centrifugation at 1.8 × 10^sup 4^ rpm for 10 min and the clarified supernatant was stored in the storage buffer as described [9].

Enzyme assay and protein determination

Recombinant protein was analyzed by SDS-PAGE [8]. The activity of the enzyme was determined by using a PCR amplification reaction with titration against a commercial Taq preparation (Roche). Human genomic DNA extracted from whole blood by DNA extraction kit (Genfanavaran) was used as template for subsequent amplification reactions. The PCR was amplified with specific product of 250bp fragment. To improve the enzyme activity, RD-buffer (Recombinant Detergent; self created name) containing Tris-HCl pH=8.8; lmg/ml Bovian Serum Albumin (BSA); mercaptoethanol O.lmM and ammonium sulfate (0.16OmM) was used in the PCR and the result was compared with standard PCR buffer.

Results

The full length of Taq polymerase gene was first PCR amplified and inserted into pTTQ vector. The recombinant plasmid was transformed into E. coli, and extracted from the cell culture. After digestion with EcoRl and San restriction enzyme, the Taq polymerase gene was gel purified and inserted into an expression vector constructed as pTTQ vector. Following the plasmid transformation, the expression of Taq polymerase was performed in 250ml in a shake flask by induction with different concentration IPTG that optimized concentration shown at 0.52mM. The induction was performed at the exponential phase. The enzyme was stored in 50% glycerol. The partial purification of the enzyme was performed through short boiling time and subsequent incubation at 75°C [2, 7-10]. The partial purified of the enzyme was monitored by SDS-PAGE (Fig. 1).

To estimate the enzyme activity, different fragments were PCR amplified with recombinant Taq. The PCR products suggested that 2µ1 of purified enzyme yields comparable results with 0.5 µl of commercial Taq polymerase, which might be the resulting from either the existence of inhibitory agents in the purified enzyme solution or a lower enzyme concentration (fig. 2).

It was also performed in a PCR with a buffer (called RD buffer) obtained improved ingredients as ammonium sulfate and BSA [6, 9, 10]. Under new conditions a 0.5µ1 of enzyme was able to yield comparable amount of PCR products as 2.5 units (0.5µ1) of commercial enzyme (Fig. 3).

Discussion

DNA polymerase from T. aquaticus has become a common reagent in molecular biology because of its utility in DNA amplification and DNA sequencing protocols [1, 6, 9, 10]. A simplified method is described here for cloning, expression and purification of recombinant Taq enzyme after overproduction in E. coli. Purification requires Ih heat-treating the E. coli lysate at 75°C, followed by centrifugation. The resulting enzyme contains a single, nearly homogeneous protein of the Taq DNA polymerase with a molecular size of 94 kDa as compared with commercial enzyme (Fig. 1). Under optimized conditions such as using a RD-buffer, 0.5µ1 of purified Taq yielded the same amount of PCR product as 2.5 units' according to 0.5 µ? of commercial enzyme (Fig. 2). The enhancing effect of RD-buffer is based on obtaining ingredients as ammonium sulfate and BSA, bind to and inactivate the putative inhibitors in the PCR [11-13].

The existence of inhibitory agents could be a consequence of sub optimized purification of enzyme [14-16] explaining the failure of PCR products with 0.5 µl recombinant enzyme (Fig. 2). On the other site, 2µ1 of recombinant Taq polymerase yielded similar amount of PCR product compared to the reaction performed by 0.5 µl commercial enzyme (Fig. 3). Grimm et al. [8] recently introduced a technique for enzyme purification known as "freezing and thawing method" that is based on rapid temperature change from -70 to -75°C, so that most of the host E. coli proteins could be denatured, and then were then easily removed from the lysate as a precipitate. This could be an alternative way to the boiling method as we have done to get more purified enzyme from cell lyses [17, 18].

Acknowledgements

The research deputy of Shahid Chamran University of Ahvaz supported this work.

References

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