Thermostable polymerases are of course key enablers of molecular biology, due to their usefulness in polymerase chain reaction (PCR). Today I am going to show you the process by which these important enzymes are isolated. You will learn how to make your own Taq polymerase using the open source expression vector pOpenTaq, as well as understand the purity grades of the commercial polymerases offered by Gene And Cell Technologies. The principles outlined in this blog post should apply to any thermostable nucleic acid polymerase.
Taq polymerase can be expressed form pOpenTaq, an open-source taq polymerase expression plasmid. pOpenTaq should be transformed into an E. coli BL21 strain and induced with 1 mM IPTG using standard protocols. Expression overnight will maximize the yield. 100% of the protein will be expressed in soluble form at 37oC.
Our purification strategy is going to be:
Lysis -> DNAse treatment -> Heat Treatment -> HPLC (optional)
A convenient way to do the lysis step is provided by Gene And Cell Technologies’ E. coli Lysis and Protein Extraction kit. This kit comes with a detailed manual taking you through the process, so we will not cover it in detail here. The Lysis process will result in a cleared lysate, which is the input material for the purification process.
DNAse is used to degrade E. coli’s DNA, which might otherwise result in false positive amplifications. After inactivating all E. coli proteins with the heat treatment step, we will have standard grade polymerase, which is free from both DNA and DNAse. An optional ion-exchange HPLC step will remove remaining oligonucleotide fragments and also serves as a redundant, extra safeguard against DNA and DNAse. The overview of each purification step and the types of molecules removed is shown in Table 1.
Table 1: Purification steps for Standard and Ultrapure Grade Taq DNA polymerase.
Standard-Grade Taq Polymerase
PCR reactions are generally very tolerant of contaminating proteins and other molecules. DNA polymerases have evolved to work inside cells, where they are necessarily surrounded by the same types of molecules that are also present in your cell lysate. There are only two mission critical contaminants that we absolutely must get rid of: DNA and DNAse.
DNA originates from E. coli’s genome, and from the expression plasmid. This DNA can cause problems relating to false amplification. This is not technically an issue when genes from a different species are amplified (e.g. human). But for work involving E. coli and common plasmids it is critical that a generally useful Taq polymerase preparation should not have this contaminant. We destroy these contaminating DNA sequences by adding the enzyme DNAse.
The DNAse itself could potentially harm your PCR reaction by degrading your template or your primers, before getting inactivated by the heating cycles. So we must remove or inactivate the DNAse completely, after it has finished its job. We will do that by playing the great strength of the Taq polymerase, its heat stability. Heating (80oC / 1 hour) will inactivate and precipitate almost all contaminating proteins, including the DNAse in a single step. The precipitate can be removed by centrifugation or filtration. And finally, remaining small molecule contaminants are removed by dialysis.
At this point, we have what we call the standard-grade Taq DNA polymerase preparation. This preparation is fit for general-use PCR, as it is free from DNA and DNAse activity detectable by PCR. The main remaining contaminant in the standard preparation consists of DNA and RNA oligonucleotide fragments too large to be removed by dialysis, but too small to affect amplifications. Some of these fragments may cause misprimings during PCR, but in practice this does not impair successful amplifications. Anyone can easily prepare their own standard-grade Taq DNA polymerase with minimal equipment, by using the open source expression plasmid pOpenTaq.
Ultrapure Grade Taq Polymerase
As we have seen, our standard grade polymerase is already free from detectable DNA and DNAse activity. With the ultra-pure preparation, we are adding a purification step using ion-exchange HPLC, which removes traces of certain remaining proteins and small nucleic acid fragments. The ion-exchange also serves as a doubly-redundant safeguard against residual DNA and DNAse contamination (if they were still present at this stage). Both our DNAse (pI = 5.2; Mw = 29) and DNA (pI < 5.0) bind more strongly to DEAE anion-exchange resin than Taq polymerase (pI = 6.0). Therefore, the standard polymerase preparation can be viewed as singly DNA and DNAse-free, while the ultra-grade preparation can be viewed as doubly (redundantly) DNA and DNAse-free.
Figure1 shows the gram-scale HPLC column we use to produce our ultrapure-grade Taq polymerase. It is not necessary to have this equipment to produce your own useful Taq polymerase free form both DNA and DNAse. We use it for the ultra-grade preparation because we can, as a double-redundant safeguard.
Figure 1: A large-scale ion-exchange HPLC column
Figure 2 shows an example of an ion-exchange gradient from one of our actual production batches of ultrapure-grade Taq polymerase at Gene And Cell Technologies. Taq polymerase was completely separated from residual proteins still present in the standard preparation, as well as short nucleic acid fragments. DNAse is no longer detectable on this gradient, as it was quantitatively removed during the heating/precipitation steps. But if we add it back at this point, it would elute together with the nucleic acid fragments, due to its more acidic isoelectric point. We always use a gradient that would theoretically remove DNAse, even when it is not present to begin with, as a redundant safeguard.
Figure 2: UV trace showing the isolation of Taq Polymerase on an ion-exchange gradient. As can be seen, the polymerase is isolated cleanly, by a wide margin, from all remaining contaminants at this point.
Optional: Omitting the DNAse step
As we have seen, DNAse is completely inactivated by the heating steps. And it is also possible (as a second layer of confidence) to separate many forms of DNAse from Taq polymerase using an appropriately desgined ion-exchange gradient. As a third layer of redundant precaution, it is possible to omit DNAse treatment entirely. In that case, it will become necessary to break the viscosity of the lysate (caused by long DNA strands) in another way.
DNAse-free methods to do this include the use of a high-pressure lysis device, high power sonication (with a probe, not a bath), or repeated syringe shearing (a powered syringe pump is highly recommeneded). These methods need to be used until the viscosity is sufficiently broken to permit downdstream purification without dramatically increasing the back-pressure. Medium-sized DNA fragments will still contaminate this kind of preparation that will give false positives in PCR amplification. It is now mandatory to remove this using ion-exchange chromatography or an equivalent highly selective DNA removal step.
At Gene And Cell Technologies, we do not currently offer a polymerase prepared in this third way. For maximum confidence we recommend our ultra-pure grade “doubly DNA & DNAse-free” preparation.
The two types of critical contaminants in DNA polymerase preparations are DNA and DNAse. DNA contamination is best tested directly by PCR. For this purpose, we use “universal” bacterial 16 ribosomal DNA primers to detect E. coli genomic DNA, and M13/pGEX primers to detect the expression plasmid, pOpenTaq. Figure 3 shows an example of this test. If these reactions come back negative, we declare the preparation “DNA free”. The standard-grade preparation will generally meet this requirement. As discussed above, we layer a redundant ion-exchange step on top of that for the ultra-pure preparation (“doubly DNA-free”).
Figure 3: Purity and sensitivity testing: PCR Reactions with 16S ribosomal DNA primers are performed with addition of increasing amounts of E. coli DNA, starting from zero. The zero sample must be blank, and then DNA-added sample must give a detectable signal down to 1 pg genomic DNA.
We detect DNAse activity by incubating a complete PCR reaction with low-concentration template (1 ng) for varying times up to 1 hour at room temperature before PCR. Then, we quantify the amount of template with Gel Green / qPCR. If no decline in signal is apparent, we deem the preparation “DNAse free”. Again the standard preparation meets this requirement, and the ultra-pure preparation uses a redudnant ion-exchange step that would remove DNAse, if it was still there at this point (“Double DNAse free”).
It is useful to express the activity of any given Taq polymerase preparation as standardized “units”. There is no general agreement on how to exactly define a unit. Many vendors’ definitions disagree with each other. At Gene And Cell Technologies, we choose an average and common approach as our unit definition:
1 unit is the amount of Taq Polymerase that incorporates 10 nmol of dNTP into acid-insoluble material in 30 min at 72°C.
If the necessary assays (e.g. radioactive) arent’ available, it is possible to use a polymerase preparation with known units as a standard. Several concentrations of polymerase should be used, and the resulting band intensities compared. Your Taq Polymerase should be diluted so that the increase of band intensity with the polymerase amount exactly parallels that of the authentic standard. Then, the unit concentration can be deemed the same (Figure 4).
Figure 4: Determination of Taq polymerase units using an authentic Taq Polymerase standard.
The optimal amount of Taq polymerase to be used per reaction varies with the amplicon length. A long PCR product (e.g. 3,000 bp) will generally work best with around 1 U of Taq pol / 50 μl reaction. When the product is much shorter (e.g. 100 bp), up to 10 U can improve the signal. That’s because the initiation of DNA replication is limiting in PCR, and a short product requires more initiation events to reach the same band intensity, than a long product.
It is possible to overuse Taq polymearse. The enzyme will autoinhibit, and cause false, smeared amplifications, if used at too a high concentration. Each unique amplification will have its own optimal Taq Polymerase concentration, which is mainly, but not exclusively, dependent on amplicon length. For most amplifications, the optimum will be around 2x to 5x wide, and it will get narrower, as the amplicon length approaches the maximum of 3-4 kb.
We hope that you will find this document useful for both expression your own Taq polymerase using pOpenTaq, as well as understanding the purity grades of Gene And Cell Technologies’ commercial Taq Polymerase preparations. If you have any questions, please ask us in the comment section below.