A novel approach for high-level expression and purification of GST-fused highly thermostable Taq DNA polymerase in Escherichia coli.
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A novel approach for high-level expression and purification of GST-fused highly thermostable Taq DNA polymerase in Escherichia coli.
Polymerases are enzymes that synthesize long chains or polymers of nucleic acids, including DNA or RNA of nucleotides. They assemble to copy the template nucleic acids DNA or RNA strand using the base-pairing interactions. One of the polymerase enzyme, Taq DNA polymerase, originally isolated from Thermus aquaticus (Taq) is an enzyme widely used in molecular biology so far.
The properties of thermostable enzymes has contributed majorly to specificity, automation, and efficacy of the polymerase chain reaction (PCR), making it a powerful tool for molecular biology research today throughout the world. Purification of Taq DNA polymerase from the original host results in low yields, labor and time consumption.
Therefore, much research has been done before to get this enzyme using an alternative host. So far, all of the existing methodology is more laborious, time consuming and requires a heavy cost. We use a new approach to purifying the enzyme with relatively high efficiency, yield and minimum time consumption using Escherichia coli (E. coli) as an alternative host.
We cloned 2500 base pairs of DNA Taq polymerase gene into pGEX-4T-1 vector containing GST-tag, downstream of tac promoter and expressed using isopropyl β-D-1-thiogalactopyranoside (IPTG) as an inducer. The enzyme was purified using chromatography efficient new approach and use in routine PCR tests in our laboratory. Our findings suggest a new approach to facilitate the availability of polymerases for molecular and diagnostic studies. In the future, may be used for purification of other recombinant peptides or proteins used in structural biology and proteomics research-based.
A novel approach for high-level expression and purification of GST-fused highly thermostable Taq DNA polymerase in Escherichia coli.
Outside Chelation: EDTA binds Closely Taq DNA Polymerase, Mutt and dUTPase and direct Inhibiting Activity dNTPase.
EDTA is commonly used as a metal ion chelator efficient enzyme cofactor. It is highly soluble, optically inactive and do not interfere with most chemicals used in making standard buffer EDTA common options for generating metal-free conditions for biochemical and biophysical investigations.
However, controversy in the literature about the activity of the metal-free enzyme is achieved by using EDTA or otherwise called our attention to the alleged effects of external EDTA chelation. Here, we show that EDTA compete for sites of hydrolases dUTPase nucleotide nucleotide binding by developing a network of interactions within the active site similar to the substrate.
To reach these findings, we apply the kinetics and molecular docking technique uses two different dUTPases. Furthermore, we directly measured the binding of EDTA to dUTPases and two other dNTPases, Taq polymerase and Mutt using isothermal titration calorimetry. EDTA binds proven exothermic and enthalpy driven primarily by submicromolar dissociation constant is much lower than with an enzyme: substrate or Mg: EDTA complex. Control of protein, including ATPase, do not interact with EDTA. Our findings indicate that EDTA can act as a selective inhibitor of the enzyme hydrolysis of dNTPs and urges a rethinking of the use of EDTA in enzymatic trial.
POLL, CT (POLL, DNA polymerase lambda, DNA polymerase beta-2, DNA polymerase kappa) (FITC)
Description: Intact Genomics T4 DNA Polymerase has both a DNA-dependent DNA polymerase activity and a potent 3´→5´ exonuclease activity. Product Includes:T4 DNA Polymerase10x T4 DNA Polymerase Buffer
Description: Intact Genomics Taq DNA Polymerase is a thermostable DNA polymerase that possesses a 5´→3´ polymerase activity (1, 2) and a 5´ flap endonuclease activity (3, 4). This product is supplied with 10x PCR reaction buffer, containing MgCl2, which produces a final Mg2+ concentration of 1.5 mM. Ideal for primary extension reaction with DNA fragments having dA overhang on 3’ ends.Product Includes:Taq DNA Polymerase10x PCR Buffer with Mg2+5x Magic Enhancer
Description: Intact Genomics Taq DNA Polymerase is a thermostable DNA polymerase that possesses a 5´→3´ polymerase activity (1, 2) and a 5´ flap endonuclease activity (3, 4). This product is supplied with 10x PCR reaction buffer, containing MgCl2, which produces a final Mg2+ concentration of 1.5 mM. Ideal for primary extension reaction with DNA fragments having dA overhang on 3’ ends.Product Includes:Taq DNA Polymerase10x PCR Buffer with Mg2+5x Magic Enhancer
Description: Intact Genomics Taq DNA Polymerase is a thermostable DNA polymerase that possesses a 5´→3´ polymerase activity (1, 2) and a 5´ flap endonuclease activity (3, 4). This product is supplied with 10x PCR reaction buffer, containing MgCl2, which produces a final Mg2+ concentration of 1.5 mM. Ideal for primary extension reaction with DNA fragments having dA overhang on 3’ ends.Product Includes:Taq DNA Polymerase10x PCR Buffer with Mg2+5x Magic Enhancer
Description: Intact Genomics Taq DNA Polymerase is a thermostable DNA polymerase that possesses a 5´→3´ polymerase activity (1, 2) and a 5´ flap endonuclease activity (3, 4). This product is supplied with 10x PCR reaction buffer, containing MgCl2, which produces a final Mg2+ concentration of 1.5 mM. Ideal for primary extension reaction with DNA fragments having dA overhang on 3’ ends.Product Includes:Taq DNA Polymerase10x PCR Buffer with Mg2+5x Magic Enhancer10mM dNTP Mix
Description: Large fragment of mesophilic B. subtilis DNA Polymerase I, which lacks the 5'->3'exonuclease activity and naturally lacks the 3'->5' exonuclease activity
Description: Large fragment of mesophilic B. subtilis DNA Polymerase I, which lacks the 5'->3'exonuclease activity and naturally lacks the 3'->5' exonuclease activity
Description: Taq DNA Polymerase is a highly thermostable DNA Polymerase that catalyzes the 5’-3’ synthesis of DNA. This polymerase has 5’-3’ exonuclease activities, lacks 3’-5’ exonuclease activity, and produces 3’-dA-tailed amplicons. PCR products made with Taq can be used with TA cloning vectors.
Description: Phi29 DNA Polymerase is a highly processive polymerase which exhibits a strong strand-displacement function. These functions allow for highly efficient isothermal amplification of circular or linear DNA templates via rolling circle amplification (RCA), multiple displacement amplification (MDA) and/or whole genome amplification (WGA). Phi29 DNA Polymerase has extremely high fidelity due to its inherent 3’→5’ exonuclease activity and can amplify from very small amounts of starting templates.
Description: The modified "hot start" enzyme which is blocked at moderate temperaturesand allows room temperature reaction setup (2.5 U/ul)
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In order to develop a test to identify the genes responsible for antibiotic resistance in gram-negative bacteria, it has been found that the standard of Taq DNA polymerase (not DNA-free) blaTEM contaminated with fragments of genes that varied in length and number. BlaTEM complete gene sequences are absent or detected in very small amounts. We developed an approach to avoid false positive findings caused by contamination in the polymerase gene sequence blaTEM conventional.
