Purification of Thermus aquaticus DNA polymerase expressed in Escherichia coli

Engelke, David R.; Krikos, Alexandra; Bruck, Mary E.; Ginsburg, David

doi:10.1016/0003-2697(90)90238-5

Cited by 148 publications

(100 citation statements)

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“…The gene for Taq DNA polymerase was PCR-amplified using the strategy and primers described by Engelke (14). The amplified product was cloned into a pTrc99A expression plasmid (Amersham Biosciences) using EcoR1 and BamH1 restriction sites.…”

Section: Methodsmentioning

confidence: 99%

Salt Dependence of DNA binding by Thermus aquaticusand Escherichia coli DNA Polymerases

Datta

LiCata

2003

Journal of Biological Chemistry

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The large fragment domains of the Type 1 DNA polymerases from Escherichia coli (Klenow) and Thermus aquaticus (Klentaq) are remarkably similar in structure (see Fig. 1), despite the fact that Taq polymerase functions at temperatures 40 -60 o higher than E. coli Pol I 1 /Klenow (1-5). A number of laboratories have been exploring the basis for the structural stability of extremophilic proteins (for a recent review, see Ref. 6). This study focuses primarily on the functional characteristics of this homologous mesophilic/thermophilic pair of polymerases in an effort to understand the functional similarities and differences between the two polymerases. Herein we focus on the initial functional step in DNA replication; that is, the binding of the polymerase to DNA and the dependence of DNA binding on salt.Taq polymerase is a single chain polypeptide with a molecular mass of 94 kDa (1-4). Due to its use in PCR, Taq polymerase is one of the most important biotechnological reagents in use in the world today. The enzyme has both polymerase activity and 5Ј nuclease activity and is a member of the Pol I polymerase family by virtue of its similarity to E. coli Pol I DNA polymerase (1-4). Like other thermophilic eubacterial DNA polymerases (5), but unlike E. coli Pol I, Taq polymerase lacks 3Ј 3 5Ј exonuclease activity (so called "proofreading activity") (1-4). Removal of the 5Ј nuclease domain from fulllength E. coli Pol I (103 kDa) produces the 68-kDa Klenow fragment (7). Removal of the same domain from Taq polymerase produces the 62.5-kDa Klentaq fragment (8).The non-covalent driving forces that lead to stable protein-DNA complexes are strongly influenced by the solution environment (salt concentration and type, temperature, pH, etc.). As a result of this dependence on the solution conditions it is impossible to understand the forces that drive these interactions based solely on structural considerations. Rather, the functional properties (thermodynamics and kinetics) of these interactions must also be investigated as a function of solution conditions to understand the origins of the stability of the complexes. Models and mechanistic explanations of function in the Pol I polymerase family are often extrapolated from one family member to another. These extrapolations are well justified given the structural similarities among the family members as revealed by the extensive series of recent co-crystal structures of different family members (for review, see Refs. 9 and 10) as well as sequence similarities among the active sites (9). Assays of function and mutagenesis studies, however, often reveal subtle and not so subtle differences among the family members (e.g. Refs. 11 and 12), but directly comparative functional studies between different family members are relatively scarce. In this study, we have characterized the basic binding equilibria of full-length Taq, Klentaq, and Klenow polymerases to DNA using a fluorescence anisotropy assay. We have further characterized the KCl and MgCl 2 dependencies of DNA binding by the dif...

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Section: Methodsmentioning

confidence: 99%

Salt Dependence of DNA binding by Thermus aquaticusand Escherichia coli DNA Polymerases

Datta

LiCata

2003

Journal of Biological Chemistry

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“…Polymerase Purification-Wild type and mutant (mutants 53, 65, 75, 94, 230, 265, 300, and 346) Taq pols were purified to homogeneity (Ͼ90% purity) using a modified procedure (17). For step 1, bacteria cultures (DH5␣ cells; 2 liters) harboring pTaq or selected mutant pTaqLIB plasmid were harvested and lysed in the presence of buffer A (30 mM Tris-HCl, pH 7.9, 50 mM glucose, 1 mM EDTA, 0.5 mM phenylmethylsulfonyl fluoride, 0.5% Tween 20, 0.5% Nonidet P-40) with lysozyme (4 mg/ml) by freezing and thawing at Ϫ70°C and 70°C.…”

Section: Methodsmentioning

confidence: 99%

Multiple Amino Acid Substitutions Allow DNA Polymerases to Synthesize RNA

Patel

Loeb

2000

Journal of Biological Chemistry

101

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DNA and RNA polymerase exhibit similarities in structures and catalytic mechanisms, suggesting that both classes of enzymes are evolutionarily related. To probe the biochemical and structure-function relationship between the two classes of polymerases, a large library (200,000 members) of mutant Thermus aquaticus DNA polymerase I (Taq pol I) was created containing random substitutions within a portion of the dNTP binding site (motif A; amino acids 605-617), and a fraction of all selected active Taq pol I (291 of 8000) was tested for the ability to incorporate successive ribonucleotides; 23 unique mutants that added rNTPs into a growing polynucleotide chain were identified and sequenced. These mutants, each containing one to four substitutions, incorporate ribonucleotides at a efficiency approaching 10 3 -fold greater than that of wild type Taq pol I. Several mutants added successive ribonucleotides and thus can catalyze the synthesis of RNA. Sequence analysis of these mutants demonstrates that at least two amino acid residues are involved in excluding ribonucleotides from the active site. Interestingly, wild type DNA polymerases from several distinct families selectively discriminate against rUTP. This study suggests that current DNA and RNA polymerases could have evolved by divergent evolution from an ancestor that shared a common mechanism for polynucleotide synthesis.Both DNA and RNA polymerase can catalyze chain elongation reaction guided by single-stranded DNA templates to generate polynucleotide products (1). The order of nucleotide addition proceeds in a 5Ј 3 3Ј direction via metal-mediated phosphoryl transfer reaction resulting in the formation of phosphodiester bond and release of pyrophosphate (2). In addition, both DNA and RNA polymerases resemble in morphology a cupped human right hand and bind DNA template and the incoming nucleotide within the active site cleft (3, 4). DNA polymerases differ from RNA polymerases in utilizing 2Ј-deoxynucleotides (dTTP, dCTP, dGTP, and dATP) rather than ribonucleotides (rUTP, rCTP, rGTP, and rATP). A detailed analysis of the polymerase active site is crucial to understanding how these polymerases distinguish between dNTPs and rNTPs, as well as to provide insights on how the two types of polymerases co-evolved to adopt similar mechanisms.Despite the similarity in protein structure and function, there is almost no sequence identity between DNA and RNA polymerases. For example, nearly all of the over 40 prokaryotic and eubacteria DNA pol Is 1 sequenced (including Thermus aquaticus pol I, Chlamydia trachomatis pol I, and Escherichia coli pol I) contain the DYSQIELR sequence within the dNTP binding site (motif A; Ref. 5), yet RNA pols only have in common the catalytically essential aspartic acid residue (6). If evolution proceeded from an "RNA world" containing RNA synthesizing enzymes to a "DNA world" with genomes replicated by DNA synthesizing enzymes (7, 8), it might be possible to gain insights into this process by substituting random sequences within the active site ...

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“…A classical purification protocol is that proposed by Engelke et al [14], combining heat pre-treatment and precipitation with polyethyleneimine, followed by ion exchange chro- . Heat-treatment of the cell lysate, as the only means of effecting Taq Pol purification, has been reported to lead to Taq Pol with a specific activity of 342 612 U/mg [32].…”

Section: Discussionmentioning

confidence: 99%

“…A classical purification protocol is that proposed by Engelke et al [14], combining heat pre-treatment and precipitation with polyethyleneimine, followed by ion exchange chro-matography, leading to Taq Pol with a specific activity of 5263 U/mg. Alternative purification protocols employ freezing and thawing of expression cell cultures (-70°C and 75°C or room temperature) [30], or ammonium sulfate precipitation [31].…”

Section: Discussionmentioning

confidence: 99%

“…DNA amplification was performed using 2.5 U Pfu Pol in 100-µL reaction mixture of PCR reaction buffer (supplied by vendor), 10 pmol of each primer, 0.2 mM of each dNTP and 50 ng of pTaq plasmid DNA. A standard PCR protocol [14] was applied to amplify the approximately 2500-bp fragment of the Taq Pol gene, using 35 amplification cycles (1 min at 94°C, 3.5 min at 72°C), and ending with incubation at 72°C for 10 min. The resulting amplification product was digested with SphI and BqlII and cloned into pQE-70 digested with SphI and BqlII.…”

Section: Construction Of the Expression Plasmid Pqetaqmentioning

confidence: 99%

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One‐step purification of Taq DNA polymerase using nucleotide‐mimetic affinity chromatography

Melissis

Labrou

Clonis

2007

Biotechnology Journal

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The thermostable Thermus aquaticus DNA polymerase (Taq Pol) has been the key factor in transforming the initial PCR method into one with huge impact in molecular biology and biotechnology. Therefore, the development of effective affinity adsorbents for the purification of of Taq Pol, as well as other DNA polymerases, attracts the attention of the enzyme manufacturers and the research laboratories. In this report we describe a simple protocol for the purification of Taq Pol from E. coli lysates, leading to enzymes of high specific activity and purity. The protocol is based on a single affinity chromatography step, featuring an immobilized ligand selected from a structure-biased combinatorial library of dNTP-mimetic synthetic ligands. The ligand library was screened for its ability to bind and purify Taq Pol from E. coli lysates. One immobilized ligand (mABSGu) of the general formula X-Trz-Y, bearing 9-aminoethylguanine (AEGu) and aniline-2-sulfonic acid (mABS) linked on the triazine scaffold (Trz), displayed the highest purifying ability. Adsorption equilibrium studies with this affinity ligand and Taq Pol determined a dissociation constant (K D ) of 0.12 mM for the respective complex, whereas ATP prevented the formation of the mABSGu-Taq Pol complex. The mABSGu affinity adsorbent was exploited in the development of a facile Taq Pol purification protocol, affording homogeneous enzyme (>99% purity, ~61 500 U/mg) in a single chromatography step. Quality control tests showed that Taq Pol purified on the mABSGu affinity adsorbent is free of nucleic acids and contaminating nuclease activities. The introduction of Taq Pol into PCR has caused this method to evolve into a widely used technique for genetic analysis [6][7][8]. Taq Pol belongs to the family of DNA polymerases I (DNA Pol I), as does the E. coli DNA polymerase I [9]. Because of its high turnover number [10], lack of proofreading activity (3'-5' exonuclease activity) [11], high temperature optimum (72-74°C), and ability to incorporate 7-deaza-3-deoxyquanosine, Taq Pol has been used extensively in DNA sequencing and other molecular biology techniques [12,13]. This enzyme holds academic interest and commercial importance and was, therefore, chosen for further evaluation of the ligand library. A successful affinity ligand selected from the library should be able to provide, in a single chromatography step and good yield, pure Taq Pol suitable for molecular biology applications. Materials and methods MaterialsThe pQE-70 vector and E. coli M15 (pREP4) strain was purchased from Qiagen (Germany). E. coli XL-1 Blue strain was purchased from Stratagene (USA). Taq Pol, Pfu Pol and deoxynucleotide triphosphates (dNTPs) were purchased from Promega (UK). The pTacTaq vector, carrying the Taq Pol coding region was a kind gift of Dr. J. F. Davidson (Department of Pathology, University of Washington, Seattle, USA). Protamine sulfate was obtained from Sigma-Aldrich (USA). Construction of the ligand libraryThe rational design and the chemical synthesis of the ligand library were...

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Purification of Thermus aquaticus DNA polymerase expressed in Escherichia coli

Cited by 148 publications

References 8 publications

Salt Dependence of DNA binding by Thermus aquaticusand Escherichia coli DNA Polymerases

Salt Dependence of DNA binding by Thermus aquaticusand Escherichia coli DNA Polymerases

Multiple Amino Acid Substitutions Allow DNA Polymerases to Synthesize RNA

One‐step purification of Taq DNA polymerase using nucleotide‐mimetic affinity chromatography

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