Abstract
Brugia malayi is one of the three causative parasites of lymphatic filariasis, a neglected parasitic disease. There is current literature suggesting that dihydrofolate reductase is a potential drug target for the elimination of B. malayi. Conditions for the expression and purification of B. malayi DHFR have not yet been investigated. As such, this study aims to establish ideal conditions to express and purify B. malayi DHFR and to determine Michaelis‐Menten kinetic values for the enzyme (kcat, Km, Vmax). The gene for B. malayi DHFR was designed, incorporating an N‐terminal His6‐tag, synthesized, and subcloned into pET15b, an E.coli expression plasmid. Expression conditions for BL21 DE3 cells were optimized at 30°C. Purification of the enzyme was completed using IMAC chromatography, eluting with 250 mM imidazole in PBS. Further work consists of optimizing protein yield, determination of kcat, Km, and Vmax and determination of Ki and IC50 values for a set of existing DHFR inhibitors such as trimethoprim and pyrimethamine, to validate B. malayi DHFR as a priority drug target.
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