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Expression and purification of a potential antifolate target, dihydrofolate reductase from B. malayi (584.7)
Author(s) -
Sanchez Karla,
Gubler Ulrich,
Siekierka John,
Goodey Nina
Publication year - 2014
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.28.1_supplement.584.7
Subject(s) - brugia malayi , dihydrofolate reductase , microbiology and biotechnology , affinity chromatography , antifolate , escherichia coli , chemistry , biology , enzyme , biochemistry , gene , filariasis , methotrexate , antimetabolite , zoology , immunology , helminths
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 (k cat , K m , V max ). 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 k cat , K m , and V max and determination of K i and IC 50 values for a set of existing DHFR inhibitors such as trimethoprim and pyrimethamine, to validate B. malayi DHFR as a priority drug target.