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Isolation of Taq Polymerase

Taq DNA polymerase is a thermostable DNA polymerase that allows for the automation of the polymerase chain reaction while simultaneously increasing both its speed and its accuracy over previous preparations. As the PCR technology has a plethora of potential uses in modern biotechnology, maintaining a sizable sample of Taq polymerase is always advantageous. As such, in this paper we show that it is possible to purify Taq polymerase from overexpressing E. coli cells in a relatively short period of time for less money than is required to purchase commercial preparations. The resultant enzyme is highly purified and is as effective as, if not more effective than, commercial preparations of the enzyme, making procedures for its purification integral to modern laboratory techniques.

Introduction

Polymerase Chain Reaction (PCR) is a critically important technology in modern molecular biology, allowing for the amplification of an initially small quantity of target DNA in a relatively short period of time. PCR has a vast range of uses in molecular technology, including but not limited to disease diagnoses, forensic sciences, and DNA sequencing. [2] The process makes use of a DNA polymerase and a series of denaturation and replication steps to amplify target DNA. By using a heat resistant DNA polymerase such as that derived from the thermophile T. aquaticus, the procedure may be automated as the polymerase will persist through the denaturation steps, removing the previous need to continuously refresh the DNA polymerase population and thereby greatly expanding the utility of this procedure. [3]

T. aquaticus DNA polymerase (Taq DNA polymerase) is active at a broad range of high temperatures with peak activity near 75°C, allowing for it to remain active even when undergoing to harsh 95°C cycles necessary to denature dsDNA during a PCR reaction. Taq polymerase lacks 3' exonuclease proofreading activity, meaning that it is able to replicate DNA very rapidly. [3] This lack of proofreading activity does make the Taq polymerase somewhat prone to errors in replication, with estimated error frequencies ranging from 2 x10-4 to 1×10-5, however these frequencies are low enough that they do not have a significant impact on biochemical analyses. [1] Results from the use of Taq polymerase are comparable to or superior than DNA polymerases which were used before the advent of this heat-stable polymerase, such as the Klenow fragment of DNA Polymerase I from E. coli. [4]

Owing to its advantages in automating the PCR process while simultaneously maintaining the integrity of copied DNA sequences, Taq DNA polymerase represents a critically important enzyme in even fairly basic laboratory procedures. As such, its purification is a beneficial tool to laboratories that frequently use this enzyme, and can be completed for a significantly lower price than buying commercial preparations. In this paper we show that Taq DNA polymerase can be purified effectively and that the resulting enzyme is at least as effective as the commercial preparation at amplifying DNA samples via PCR.

Materials and Methods

Strain Culture and Inoculation

Taq DNA polymerase overproducing E. coli were inoculated in 500mL Luria Broth containing ampicillin incubated in a shaking incubator at 37°C. After one hour 500µL 0.5M IPTG was added to induce Taq DNA polymerase production. Growth was monitored by regularly collecting samples, the absorbance of which was measured in a spectrophotometer at 650nm, producing a growth curve (Fig 1A). Twenty six hours after inoculation, the samples were collected by centrifuging at 6000rpm for 20 minutes and discarding the supernatant.

Protein Assay

All protein assays used to establish protein quantity conformed to the procedures of the Bio-Rad DC protein assay. Briefly, an 8-part Bovine Serum Albumin (BSA) standard dilution series was created with concentrations from 2000µg/mL to 25µg/mL in the buffer of interest. Working Reagent A – an alkaline copper tartrate solution containing 20µL/mL SDS – was added in 500µL aliquots to 100µL aliquots of the BSA standards and the unknown samples, all of which were then vortexed briefly. Reagent B – a dilute Folin reagent – was then added in 4mL aliquots to each sample, which were then allowed to sit for 15 minutes. A spectrophotometer was then blanked at 750nm, and the absorbance of 1mL of each sample was recorded to generate a standard curve to establish unknown protein concentrations.

Confirmation of Induction

A 1mL aliquot of the induced E. coli was resuspended in 250µL dH2O, and 10µL and 30µL aliquots of induced and uninduced sample were diluted to 100µL with dH2O and were assayed with the Bio-Rad DC protein assay as previously described. The remainder of the 1mL aliquots were precipitated with 200µL 10% trichloracetic acid (TCA) to eliminate sample viscosity, centrifuged, and resuspended in 100uL of 1X gel sample buffer. Next, 10µg of the uninduced and induced samples in sample gel buffer were added to consecutive wells in an SDS-PAGE gel, as were 10µL of molecular weight markers. The gel was run for 15 minutes at 150V and then at 200V until the dye front reached the gel edge. The gel was stained in Coomassie Blue R-250 stain. The resulting gel was then analyzed to confirm induction (Fig 1B).

Heat Precipitation

The remaining E. coli were lysed by resuspending them in 10mL of Buffer A, consisting of 50 mM Tris-HCl, pH 7.9, 50 mM dextrose, 1mM EDTA with 4mg/mL lysozyme and incubated at room temperature for 15 minutes. Next, 10mL of Buffer B containing 10 mM Tris-HCl, pH 7.9, 50 mM KCl, 1mM EDTA, 0.5% Tween 20, 0.5% NP-40 and 200uL PMSF was added and incubated in a 75oC water bath for 1 hour. The sample was then pelleted at 8000rpm for 15 min at 4oC and the supernatant containing the Taq DNA polymerase was saved. A 200µL aliquot was stored for later use.

Ammonium Sulphate Precipitation, Ultrafiltration, and Dialysis

The remaining proteins were precipitated with 516g/mL ammonium sulphate to achieve 80% saturation, and were stirred on ice for 30 minutes. The precipitate was then pelleted at 9000rpm for 10 minutes at 4oC and resuspended in Buffer C, consisting of 20 mM HEPES, pH 7.9, 1 mM EDTA, 0.5% Tween 20, 0.5% NP-40 with 50mM KCl. A 200µL aliquot was stored for later use. Next, the sample was then loaded into a concentrator device with a molecular weight cutoff of 30kDa and spun for 40 min at 5,000rpm so that the sample volume was reduced to 2mL. The sample was then dialyzed against 1L of Buffer C containing 50mM KCl.

Ion Exchange Chromatography

BioRex 70 resin was equilibrated until it reached a pH of 7.9 and packed in a column at a rate of 1mL/min with a final column volume of 12mL. The column was then washed with 24mL of Buffer C containing protease inhibitors. The sample was then loaded and flowed at a rate of 1mL/min while the column was washed with 60mL Buffer C with 50mM KCl containing PMSF, with 5mL fractions being saved. The column was then eluted with 60mL Buffer C with 200mM KCl and stored as 2mL fractions.

Confirmation of Elution

Protein elution was confirmed by performing a Bio-Rad DC protein assay on the samples and plotting the absorbance versus the elution fraction number. An SDS-PAGE analysis of the samples was also performed as follows. A 12.5µL aliquot of each fraction was mixed with 2.5μL of 6X sample buffer and incubated on a dry heat block at 90°C for 3 minutes. The samples were then loaded into a gel with 10µL molecular weight markers and run for 15 minutes at 150V and then at 200V until the dye front had migrated to the bottom of the gel. The gel was stained with Coomassie Blue R-250 and was analyzed to determine which fractions contained Taq DNA Polymerase (Fig 2). The fractions containing the most protein were pooled together in a concentrator device with a molecular weight cutoff of 20kDa and were centrifuged at 5000rpm for approximately 20 minutes until the sample volume was reduced to 2mL. The sample was then dialyzed against 500mL Storage Buffer containing 20mM HEPES pH 7.9, 1mM EDTA, 100mM KCl, 1mM dithiothreitol, 50% glycerol for 48 hours.

Assessment of Purification by SDS-PAGE and Preparation for Western Blot

A Bio-Rad DC protein assay was used to generate a standard curve, which was used to establish the amounts of protein in four samples from various points in the purification process – after heat precipitation, after ammonium sulfate precipitation and dialysis, after BioRex 70 elution, and after concentration in storage buffer. Next, 10µg of each sample and 2µL Taq DNA Polymerase standard were each combined with 2.5μL of 6X sample buffer and diluted to 15µL with dH2O. The samples and standard were then loaded into a gel in duplicate along with 10µL molecular weight markers and run for 15 minutes at 150V and then at 200V until the dye front reached the bottom of the gel. The gel was then split in half, with one half stained in Coomassie Blue R-250 (Fig 3A) and the other half was stored in transfer buffer for 10 minutes and was then underwent electrophoretic transfer as follows. The gel was assembled between filter paper, a PVDF membrane, and filter paper and transferred for 30 minutes at 10V.

Western Blotting

A Western Blot of the PVDF membrane was performed as follows. First the membrane was blocked for 30 minutes in 30mL TTBS containing 5% milk. Next it was incubated with 10mL of 1:20,000 anti-Taq DNA polymerase (purchased from Antibody Research Corporation) which served as the primary antibody. It was then washed for 7 minutes in fresh TTBS containing 5% milk three times and incubated in 10mL of 1:20,000 anti-mouse IgG (provided in a Vectastain ABC-AmP Kit from Vector Laboratories) which served as a secondary antibody. The membrane was subsequently washed three times in fresh TTBS for 7 minutes, incubated with 10mL ABC-Amp reagent for five minutes, and washed three times in fresh TTBS for 4 minutes. The sample was then equilibrated in 10mL of 0.1M Tris, pH 9.5 for 5 minutes before 10mL the color development solution - BCIP/NBT – was added until bands began to appear. The reaction was terminated with 30mL 10mM EDTA for 4 minutes, and the gel was rinsed in dH2O and allowed to dry (Fig 3B)

Polymerase Chain Reaction

To test the efficacy of the purified Taq DNA Polymerase sample, a PCR was run on five different samples of a 1:1 Taq dilution, a 1:5 Taq dilution, a 1:20 Taq dilution, a commercial Taq preparation (as a positive control), and a blank (as a negative control), all diluted in dilution buffer. A master mix consisting of 15µL 10X Buffer, 18µL MgCl2, 6µL dNTPs, 7.5µL M13R primer, 7.5µL M13F primer, 3µL template DNA, and 18µL dH2O was created, and 24.5µL was combined with 0.5µL of each Taq sample. The samples were then placed in the thermocycler and collected after the reaction had completed.

Analysis of PCR Products

To visualize the PCR products, the samples underwent gel electrophoresis through a 2.0% agarose gel in Tris-Boric acid-EDTA, pH 8.0. The five 25μL samples were mixed with 5μL 6X loading dye run for 40 minutes at 100V as were 20μL of DNA molecular weight ladder. The gel was then photographed with UV light and the resulting gel was analyzed to determine the molecular weight of the PCR products (Fig 4).

Results

The initial steps of these purification procedures were carried out without incident, with the E. coli cells being induced to produce Taq DNA polymerase early in the exponential phase of growth (Fig 1A). The cells were collected after 26 hours of growth and an SDS-PAGE analysis confirmed the successful induction of Taq DNA polymerase (Fig 1B), with the indicated protein having a calculated weight of 103kDa versus the actual weight of Taq polymerase, which is 94kDa. These values are very close to one another, indicating that this protein is presumably Taq polymerase, confirming successful induction.

Since the induction procedures were successful, the cell lysates underwent heat precipitation to denature the majority of non-Taq DNA Polymerase proteins, as while Taq is heat stable, the majority of proteins cannot survive such harsh conditions and are consequently precipitated out of solution. The sample was then precipitated with 80% ammonium sulfate and underwent ultrafiltration and dialysis against Buffer C containing 50mM Kcl in order to remove the salts from the solution so that the sample could successfully undergo ion exchange chromatography.

The sample was next eluted through a Biorex 70 column by means of ion exchange chromatography. Though 2mL fractions of the elulate were meant to be collected, issues with the vacuum pump arose which resulted in only one fraction being successfully recovered. Nonetheless, this fraction contained a significant amount of Taq polymerase, with the protein band having a calculated weight of 102.7kDa, which is sufficiently close to the actual weight of Taq polymerase (94kDa) as to suggest that this protein is indeed Taq polymerase (Fig 2), and that there is little to no contaminating protein. This final purified sample was then dialyzed against a glycerol-containing buffer for long term storage.

After the purification process was complete, and SDS-PAGE analysis of four different time points over the course of the purification was performed (Fig 3A). This gel clearly demonstrates the progressive purification of Taq polymerase, with the final sample having virtually no contaminating protein as compared to the earlier samples which have a significant amount of contaminant. For uncertain reasons, the post elution sample (CE) did not contain any protein. This is likely due to sample mislabeling following the confusion due to mechanical issues with the vacuum pump during column elution, and does not reflect upon the ultimate purification of Taq polymerase, as the sample in storage buffer is demonstrative of a successfully purified protein.

A Western blot of the sample was also performed (Fig 3B) which demonstrates that the purified protein is indeed Taq polymerase. The reason for a lack of evident Taq polymerase in the early samples (HP, AS) is uncertain, but likely stems from a relatively lower amount of Taq present in the sample. As Taq DNA polymerase constituted a lower percent of the sample, it may have had more competition to bind the primary antibody resulting in lower binding and a less prominent signal. This lack of binding is not indicative of a lack of Taq in these samples, as the protein is clearly evident in later samples meaning it must have been present in these samples as well.

An analysis of the protein amounts over the course of the purification procedures was performed (Table 1). This analysis indicates that, as expected, as the Taq was increasingly purified the amount of overall protein in the sample decreased significantly, from initial values of 6532µg after heat purification to 1275µg after final concentration. The increase between column elution and final concentration is likely a result of slight errors in the protein assay, and does not reflect an increase in Taq polymerase at this time point.

Finally, to test the functionality of the purified Taq polymerase, a series of purified Taq dilutions were prepared and used to perform a PCR to amplify a ~200bp sample strand of DNA. A commercial Taq standard was also included against which to compare the results. After the PCR was complete, the products underwent gel electrophoresis in a 2% agarose gel (Fig 4). This gel shows that all four Taq polymerase samples successfully replicated the target DNA strand, with each replicated DNA strand having a calculated molecular weight of 202kDa or 216kDa, very close to the expected 200kDa (Table 2). All four samples expressed a high amount of replicated contaminant DNA evident at the top of the gel, likely due to environmental contamination due to the assay's high levels of sensitivity. Nonetheless, these results clearly demonstrate the ability to successfully purify Taq DNA polymerase, with the purified sample being at least as effective as the commercial preparation if not more so, even at a 1:20 dilution of the enzyme.

MWM = Molecular Weight Markers, UN = Uninduced, IND = Induced

Fig 1. A. Growth curve for Taq DNA polymerase expressing E. coli. Growth measured by recording sample absorbance at 650nm as a function of time for a 26 hour period. The arrow indicates the time at which sample was induced with IPTG to activate the lac operon of the cells, resulting in overproduction of Taq DNA Polymerase. Cells were collected at the 26 hour time point in the stationary phase of growth. B. Confirmation of induction. This SDS-PAGE gel contains samples of both induced and uninduced E. coli. The band present in the induced sample but not in the uninduced sample (indicated by the arrow) has a calculated molecular weight of approximately 103kDa, whereas Taq DNA polymerase has an actual weight of 94kDa.

  • HP= Post Heat Precipitation, AS= Post Ammonium Sulfate Concentration, CE= Post Column Elution, FC= Final Concentration,

CT= Commercial Taq Preparation, MWM= Molecular Weight Markers

Fig 3. A. Purification Scheme. This SDS-PAGE gel constitutes an overview of the purification process for Taq DNA polymerase. The gel contains 10µg protein samples of different time points in the purification process as well as commercial Taq DNA polymerase. B. Confirmation of Purification. This Western Immunoblot utilized an anti-Taq DNA polymerase as a primary antibody and Biotin-linked anti-IgG as a secondary antibody. The blot was developed using BCIP/NBT and the reaction was terminated with EDTA to prevent over-development.

1= 50bp Ladder Markers, 2= 1:1 Taq Dilution, 3= 1:5 Taq Dilution, 4= 1:20 Taq Dilution, 5= Commercial Taq, 6= No Taq

Fig 4. Analysis of PCR Products. This 2.0% agarose gel was loaded with 30µL of the PCR products and run for 40 minutes at 100V. The image was photographed under ultraviolet light, with nucleic acid bands fluorescing. The amplified DNA is evident near the bottom of the gel, while contaminating DNA is amplified at the top.

Table 1. Protein quantities of Purification Process. This table contains the volumes, concentrations, and protein quantification of the four samples analyzed in Fig 3 from different points in the purification process. Concentrations were established by performing a Bio-Rad DC protein assay, and protein amounts were calculated by multiplying sample concentration by sample volume.

Table 2. Analysis of PCR Products. The calculated values in this table were generated by measuring the migration distances of the bp ladder bands in the agarose gel (Fig 4). The migration distances were then graphed against the base pair values and a line of best fit was generated (R2=1), to which the sample bands were correlated. The approximated values of DNA fragment size are summarized above.

Discussion

Taq DNA polymerase is well established as a highly useful heat stable enzyme which can successfully copy DNA with a fairly high degree of fidelity, amplifying small amounts of DNA in the harsh denaturing conditions mandated by the polymerase chain reaction. This series of experiments has demonstrated the relatively straightforward and successful purification of Taq DNA polymerase, illustrating that it may successfully be completed within a 24-48 hour period once the E. coli cells have been collected.

As discussed in the results section, the final concentration of purified Taq DNA polymerase is very pure, as is demonstrated in the purification scheme (Fig 3A). The purified enzyme amplifies a sample DNA sequence as well as or better than the commercial Taq preparation, even at a 1:20 dilution, (Fig 4)with the amplified sequence remaining effectively the same in terms of its length (Table 2). While there does appear to be more background DNA amplification in the purified Taq polymerase than in the commercial preparation, this is likely due to contamination with external DNA as the assay is highly sensitive, although it is possible that the contaminant consists of aggregates of improperly replicated DNA.

As previously mentioned, a problem arose with the vacuum pump during the column elution step which resulted in the loss of a large portion of the eluate, but not the portion containing Taq polymerase. While this sample may ultimately have been slightly less concentrated than intended, and though this issue prevented the creation of an elution profile, the overall effect on the purification procedures was negligible, as the final enzyme sample was fully functional and highly purified, containing minimal levels of contaminating protein content.

While Taq DNA polymerase is very effective for rapid amplification of a target DNA sequence, there are some problems with its activity. For one, despite careful primer design, off target DNA amplification may occur at lower temperatures during a PCR. To reduce this risk and increase the specificity of this assay, a “hot start” technique may be used. These techniques block the primer extension under low temperatures conditions, whether by physical separation, DNA polymerase inhibition, or modified primer constructs. [5] Another fault with Taq DNA polymerase is that it lacks 3'-5' exonuclease activity, as previously mentioned. While the error rate is still relatively low, DNA sequencing benefits from a minimal number of amplification errors. To this effect, other thermostable DNA polymerases have been purified from hyperthermophiles. Pfu DNA polymerase is one such enzyme, and it contains 3'-5' exonuclease activity yielding an error rate 10-fold lower than that of Taq polymerase. [6]

While alternatives to Taq polymerase exist and are associated with certain advantages, Taq remains a relatively inexpensive and easy to purify enzyme which is able to rapidly replicate DNA with a fairly high fidelity, making simple purification techniques such as those demonstrated in this paper invaluable in modern laboratories.

References

  • Eckert, KA and TA Kunkel (1991). DNA polymerase fidelity and the polymerase chain reaction. Genome Research. 1:17-24.
  • Vosberg, HP (1989). The polymerase chain reaction: An improved method for analysis of nucleic acids. Human Genetics. 83:1-15.
  • Innis, MA et al. (1988). DNA sequencing with Thermus aquaticus DNA polymerase and direct sequencing of polymerase chain reaction-amplified DNA. Biochemistry. 85:9436-44.
  • Keohavong P and WG Thilly (1989). Fidelity of DNA polymerases in DNA amplification. Biochemistry. 86:9253-7.
  • Lebedev AV, et al. (2008) Hot Start PCR with heat-activatable primers: a novel approach for improved PCR performance. Nucleic Acids Research. 36.
  • Cline JC, Braman JC, and Hogrefe HH (1996). PCR fidelity of Pfu DNA polymerase and other thermostable DNA polymerases. Nucleic Acids Research. 24:3546-51.

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