In-house expression and purification of phusion DNA polymerase for high fidelity polymerase chain reaction. (pcr) amplification

Abstract

Background: DNA polymerases are indispensable tools in the molecular biology field during synthesis of DNA molecules in the Polymerase Chain Reaction (PCR). Phusion DNA polymerase engineered from a processivity enhancing domain and Pfu DNA polymerase possesses a high extension speed and a 3´ to 5´ exonuclease (proof-reading) activity and hence exhibits an extremely low error rate in PCR amplification. However, purchasing processed Phusion DNA polymerase from the manufacturer is expensive. Objectives: The aim of this study was to express and purify Phusion DNA polymerase in-house using simplified cost-effective purification methods. Materials and Methods: The E. coli BL21 (DE3) strain with the Phusion gene was gifted by CEND University of California and San Francisco. The cloned gene sequence for Phusion polymerase was over-expressed in E. coli BL21 (DE3) under IPTG induction. Upon rupturing the cell membranes, the bacterial lysate in which the Phusion DNA polymerase was contained was heated at 75°C for 1 hour. Due to its thermostability at high temperatures, Phusion DNA polymerase, remained intact in supernatant while most of proteins were denatured and precipitated out. Cation exchange chromatography and dialysis were further employed to separate the Phusion polymerase from the other proteins. To assess its functionality, the purified Phusion DNA polymerase was used in PCR to amplify the UGT1A1 gene and rpoB gene responsible for rifampicin resistance in M. tuberculosis. Results: Over expression of Phusion polymerase in E. coli BL21 (DE3) strains was successful after overnight incubation with IPTG and a band of relatively 90 kDa in Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis (SDS-PAGE) was obtained. The Agarose Gel Electrophoresis of the UGT1A1 gene and rpoB gene amplicons produced bands with a molecular weight of approximately 243 bp and 543 bp respectively using the purified Phusion polymerase. Conclusion: The Phusion DNA polymerase produced in-house successfully and therefore can be used in day-to-day amplification where high-fidelity is required such as in DNA sequencing and cloning to improve on the quality of results obtained. Keywords: Protein expression, Protein purification, Phusion DNA polymerase, Pyrococcus furiosus, Polymerase chain reaction