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Expression And Purification of A Potential Antifolate Target, Dihydrofolate Reductase from B. malayi
Author(s) -
Sanchez Karla,
Gubler Ueli,
Siekierka John,
Goodey Nina
Publication year - 2015
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.29.1_supplement.721.34
Subject(s) - brugia malayi , dihydrofolate reductase , biology , antifolate , microbiology and biotechnology , plasmid , gene , biochemistry , genetics , filariasis , antimetabolite , chemotherapy , zoology , helminths
Brugia malayi is one of the three causative parasites of lymphatic filariasis, or elephantiasis, a parasitic disease causing swelling of extremities and lymphatic damage. Current literature shows antifolate drugs inhibit B. malayi , suggesting that dihydrofolate reductase (DHFR) is a potential drug target for the elimination of the parasite. Conditions for the expression and purification of B. malayi DHFR have not previously been investigated. As such, this study aims to establish ideal conditions for the expression and purification of Bm‐ DHFR, to determine kinetic values for the enzyme (k cat , K m , V max ), and to study existing antifolate compounds as possible inhibitors. Initial work was completed using a Bm ‐DHFR gene whose sequence was obtained from the UniProt database (accession number A8QGA9). When compared to the Bm ‐DHFR sequence entry from Wormbase (BM34009), the UniProt sequence was found to be missing a loop known to play a role in catalysis (FG loop) consisting of residues #113‐125 in BM34009. The Uniprot Bm ‐DHFR was difficult to express in soluble form and exhibited no catalytic activity. These findings correlate with previous studies showing that point mutations in the E. coli DHFR FG loop result in markedly decreased catalytic activity. A new Bm ‐DHFR construct is currently being used with the sequence obtained from BM34009 entry. This Bm ‐DHFR was designed incorporating an N‐terminal His6‐tag and was obtained in the pUC57 vector. Current work includes subcloning the gene into pET25b an E. coli expression plasmid. Expression and purification conditions will be optimized and K i and IC 50 values will be determined for a set of DHFR inhibitors including trimethoprim and pyrimethamine to validate Bm‐ DHFR as a priority drug target.