Expression and purification of phusion DNA polymerase from Escherichia coli BL21-DE3 at the molecular biology laboratory of Makerere university biomedical research center

Abstract

Background Polymerase Chain Reaction (PCR) based assays are considered a gold standard diagnostic tool with broad application in genetic testing (Chau et al., 2020). Thermostable DNA polymerases are particularly important in molecular biology for PCR-related techniques (Ishino & Ishino, 2014). For low resource settings however, procurement of commercially available DNA polymerases has proved costly and necessitated the need to cut costs where in-house production of these enzymes could be a possible solution. In this experiment therefore, we expressed and purified Phusion DNA Polymerase from Escherichia coli BL21 (DE3) cells containing plasmid and further used the purified protein to perform PCR assays. Methods BL21 DE3 cells containing the Phusion DNA Polymerase Plasmid were cultured in Luria Bertani (LB) media with Chloramphenicol (34µg/mL) and Kanamycin (50µg/mL) antibiotics to an OD600 of 0.4. The cells were then induced using Isopropyl β-d-1-thiogalactopyranoside (IPTG) overnight, harvested and purified using physical and chemical properties of Phusion DNA polymerase via heat separation and Cation exchange chromatography. The protein was concentrated by dialysis method. Polymerase Chain reaction (PCR) assays were used to validate the activity of the enzyme produced. Results High Fidelity Phusion DNA was purified and most of it was collected in the 35% high salt buffer C cation exchange flow through. The different serial dilutions of the concentrated enzyme after dialysis successfully amplified UGT1 gene until the 1:8 dilution. The 1:4 dilution gave the best amplification and was further used to amplify InhA gene in archived Mycobacterium tuberculosis isolates at the Laboratory. Conclusion In-house production and purification of recombinant proteins is possible and could help facilities in low resource settings cut costs on procurement and pipeline of reagents