Expression, purification and characterization of high-fidelity DNA polymerase

Abstract

DNA polymerases found in all living cells discovered to date are the enzymes that synthesizes a new DNA strand complementary to template single stranded DNA. These enzymes do not only play a major role in the transmission of genetic information across generations during cell division, they also form the basis of Polymerase Chain Reaction (PCR), which is one of the most important in-vitro diagnostic techniques today. In addition to synthesis ability, DNA Polymerases may also have other properties including processivity, which is known as the ability of continuous polymerization, fidelity, which is known as the synthesis accuracy, and nucleotide selectivity. Thermostable DNA polymerase enzymes are mostly preferred in PCR-based studies because it is high importance that the stability of the enzymes used do not decrease depending on temperature. Taq DNA polymerase is the first discovered polymerase, which is a well-known enzyme used in a wide range of applications. Following the discovery of Taq DNA polymerase, the high-fidelity DNA polymerase was discovered in 1991 as a highly thermophilic DNA polymerase. Due to its high thermostability and proofreading properties, high-fidelity DNA polymerase is widely used in the applications that require high accuracy such as molecular cloning. High-fidelity DNA polymerase is an enzyme with a length of 775 amino acids and a molecular weight of about 90 kDa. This enzyme can perform 3'-5' exonuclease (proofreading) activity, which allows the addition of the correct nucleotides by removing the wrong nucleotides added to the structure during DNA synthesis. Due to this feature, it reduces the error rate during synthesis (1.3×10-6 mutations/base pairs/duplications), resulting in about 8 times less errors compared to Taq DNA polymerase. Many researchers have produced this protein by cloning it from Pyrococcus furiosus, a hyperthemophilic archaea, into different strains of Escherichia coli. The purification step is simplified by adding an affinity tag to the N- or C-terminus during the cloning. Based on these tags and various biophysical properties of the protein, purification protocols were created by affinity chromatography or ion exchange chromatography. In this study, we aimed to purify and characterize the high-fidelity DNA polymerase enzyme by taking the advantage of its thermal stability and 10X Polyhistidine-tag after bacterial production with high efficiency and low cost. For this purpose, commercially purchased pET16B. High-fidelity polymerase's plasmid DNA with a 10X Polyhistidine-tag at the N-terminus was used. The plasmid pET16B. High-fidelity polymerase was transformed into competent E. coli BL21(DE3) cells containing GroEL/GroES chaperonins to ensure soluble expression of the protein. In the first step of purification of High-fidelity DNA polymerase, which is a thermostable protein, all the folded proteins obtained from bacterial cells were heated and centrifuged to separate impurities with less thermal stability. High-fidelity DNA polymerase in soluble form was purified using IMAC affinity chromatography. The pure product was taken into a storage buffer containing 50% glycerol by filtration. The GroEL/GroES chaperonin system is a system that enables unfolded proteins with a molecular weight of 2-100 kDa to be folded in vitro and in vivo. Given that GroEL/GroES system can increase the folding of co-expressed recombinant proteins of different sizes by up to 70%, this system was employed in the production of High-fidelity DNA polymerase. However, while this system increases the amount of target protein, it can also increase the amount of impurities. Therefore, the purification of High-fidelity DNA polymerase was highly challenging. So that, various buffer compositions were used in order to optimize one step IMAC purification. Co-expression system was induced using IPTG for expression of pET16B. Expression of the polymerase regulated by the Lac operon. Since the growth temperature was chosen in the range of 12-20°C, where the metabolic rate of the cell and thus the growth rate was selected, the amount of protein folded by the chaperones was increased. Most of the impurities that increased with the target protein were eliminated with the 90°C heat treatment step. While heat sensitive proteins are eliminated from the environment, thermostable high-fidelity DNA polymerase enzyme, GroEL, and GroES proteins are still present. The separation of GroEL and GroES was achieved by applying IMAC affinity chromatography to increase the purity of the high-fidelity DNA polymerase enzyme. Purification results were analyzed by SDS-PAGE and immunoblotting methods. At the end of 500 ml bacterial production and purification process with three biological repetitions, high-fidelity DNA polymerase enzyme of similar quality with its commercial counterparts was produced, which can be used for a total of 60 000 PCR reactions with ~90% purity. The protein band on the SDS-PAGE gel was excised and analysed by peptide mapping using Liquid Chromatography-Mass Spectrometry (LC-MS) system to confirm that the produced protein is the target protein. According to the analysis, it was concluded that the purified protein was the target DNA polymerase. In order to determine if the purified protein is correctly folded or not, the secondary structure analysis of the protein with a purity over 90% was performed using Circular Dichroism (CD) in the far-UV (<260 nm) range. As a result of the study, the protein showed an apparent α-helix secondary structure with two minima at 208 and 222 nm wavelengths and a maximum at 190 nm wavelengths. Functional analysis of the protein on its folded state was completed by performing the Polymerase Chain Reaction (PCR). The 825 bp DNA region with 49.6% G-C content, and 1947 bp DNA region with 61% G-C content was amplified. These regions were selected considering the processivity of the enzyme. No-template control was used as negative control. The amplified regions were analyzed comparatively with commercial enzymes and it was observed that the target regions were successfully amplified. Commercially available high-fidelity DNA polymerase enzymes do not have endonuclease contamination and exonuclease contamination. Within the scope of quality control experiments, both endonuclease and exonuclease contamination of three biological replicates of our high-fidelity DNA polymerases were compared with commertial ones. λ DNA and λ DNA digested with HindIII were used as positive control. As a result, it has been shown that commercial enzymes and our high-fidelity DNA polymerase enzyme do not have endonuclease and exonuclease contamination. Another important test for demonstrating the quality of commercial enzymes is testing whether the protein remains stable under different conditions. For testing the stability of the polymerase, the effect of freeze-thaw stress repeated 20 times and also the effect of incubation of the enzyme for 5 days at room temperature were evaluated. As a result of these experiments, it has been shown that the freeze-thaw process during the general use of purified enzyme does not cause a negative effect on the activity of the protein, and that if the purified enzyme is forgotten at room temperature for up to 5 days during use, they can polymerize without reducing their activity. In conclusion, with this study, we have produced high-fidelity DNA polymerase with the same stability and processivity as commercial polymerases produced by large biotechnology companies with high efficiency and purity.