Expression and purification of phusion polymerase from BL21 (DE3) cells of Escherichia Coli B strains at the Genomics, Molecular and Immunoloy laboratory at Makerere University College of Health Sciences.
Ssemuyaba, Joshua Vitali
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Phusion polymerase enzyme is a combination of a proofreading polymerase and a processivity-enhancing domain developed by Finn zymes, resulting in one of the most reliable and robust polymerases available (Westburg, 2021). The Phusion DNA polymerase can be used for high-fidelity PCR, cloning, and other applications. Because of its proof-reading activities, it generates templates for sequencing. Because it is highly heat stable and can endure temperatures of up to 115°C, it is ideal for amplification of problematic (GC-rich) templates. Due to the presence of a "clump," long-range PCR (up to 20 kb) is possible (Thermofisher, 2021). This study was aimed at the expression of Phusion polymerase from BL21 (DE3) cells of Escherichia Coli B strains. In this study, the functionality of Phusion Polymerase was also tested. The E. coli BL21(DE3) Phusion pLysS cells were provided by CEND University of California and San Francisco. Pipettes, molecular reagents, cation exchange chromatography filters, PCR machine, Centrifuge, Belly dancer, Weighing Scale, Gel apparatus, Incubator, Water bath, Culture plates, Gloves, Falcon tubes, Eppendorf tubes were provided by GMI laboratory. The DNA was also provided by the GMI laboratory. The forward and reverse primers, the positive and negative controls as well as the UGT1A1 gene were provided by the GMI laboratory. The cells were cultured on LB broth. Protein purification was done by cation exchange chromatography and dialysis. The protein was then subjected to an SDS-PAGE. This was followed by dilution and a PCR was set up for these dilutions. This was followed by gel electrophoresis to visualize the results. The protein was also used in a PCR to amplify the GyrA gene and this was also successful after results were visualized when gel electrophoresis was done. Growth of cells after culture was successful and overexpression also happened after induction with IPTG. The protein remained intact after heating at 75°C and purification after cation exchange chromatography and dialysis was successful. Phusion polymerase was present after performing an SDS-PAGE. After dilution and performing a PCR run while targeting the UGT1A1 gene, it was found that the protein was functional after amplification of this gene was confirmed after performing gel electrophoresis. This was further proven after using the protein to amplify GyrA in another PCR run. Expression and purification of Phusion polymerase in-house were successful and the enzyme that was produced can be used in PCR amplification where high fidelity is required.