Expression and purification of dihydrofolate reductase from Wuchereria bancrofti as a potential antifolate drug target

Presentation Type

Abstract

Faculty Advisor

Nina Goodey

Access Type

Event

Start Date

25-4-2025 9:00 AM

End Date

25-4-2025 9:59 AM

Description

Elephantiasis, also known as lymphatic filariasis, affects around a billion people in 54 countries and remains a significant worldwide health concern. Wuchereria bancrofti (Wb), the parasitic worm responsible for filariasis, depends on Dihydrofolate reductase (DHFR), the enzyme that catalyzes the conversion of dihydrofolate to tetrahydrofolate for DNA replication and cell division. DHFR is an important drug target in the treatment of cancer and bacterial infections. Due to DHFR’s central role in folate-mediated metabolism and necessity for DNA synthesis, it is a major focus in this study for the identification of potential inhibitors that could disrupt the survival and replication of Wb. An N-terminal His6-tagged construct was used to produce the enzyme in E. coli, and isopropyl β-D-thiogalactopyranoside (IPTG) was used to induce the production of the Wb DHFR enzyme. Methotrexate -agarose resin and nickel-nitriloacetic acid (Ni-NTA) were used in a two-step affinity chromatography method to purify the enzyme. To test for the presence and concentration of the purified protein, a Nanodrop at 280 nm was used to measure the protein yield. We will analyze the isolated enzyme's Michaelis-Menten kinetic parameters, KM (μM) as a substrate affinity measure and kcat (s⁻¹) as the catalytic turnover rate, to gain a better understanding of the Wb DHFR active site. This research lays the foundation for targeted inhibitor screening by identifying selective antifolate compounds that could lead to effective treatments for lymphatic filariasis, potentially improving the lives of millions affected by this neglected tropical disease.

Comments

Poster presentation at the 2025 Student Research Symposium.

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Apr 25th, 9:00 AM Apr 25th, 9:59 AM

Expression and purification of dihydrofolate reductase from Wuchereria bancrofti as a potential antifolate drug target

Elephantiasis, also known as lymphatic filariasis, affects around a billion people in 54 countries and remains a significant worldwide health concern. Wuchereria bancrofti (Wb), the parasitic worm responsible for filariasis, depends on Dihydrofolate reductase (DHFR), the enzyme that catalyzes the conversion of dihydrofolate to tetrahydrofolate for DNA replication and cell division. DHFR is an important drug target in the treatment of cancer and bacterial infections. Due to DHFR’s central role in folate-mediated metabolism and necessity for DNA synthesis, it is a major focus in this study for the identification of potential inhibitors that could disrupt the survival and replication of Wb. An N-terminal His6-tagged construct was used to produce the enzyme in E. coli, and isopropyl β-D-thiogalactopyranoside (IPTG) was used to induce the production of the Wb DHFR enzyme. Methotrexate -agarose resin and nickel-nitriloacetic acid (Ni-NTA) were used in a two-step affinity chromatography method to purify the enzyme. To test for the presence and concentration of the purified protein, a Nanodrop at 280 nm was used to measure the protein yield. We will analyze the isolated enzyme's Michaelis-Menten kinetic parameters, KM (μM) as a substrate affinity measure and kcat (s⁻¹) as the catalytic turnover rate, to gain a better understanding of the Wb DHFR active site. This research lays the foundation for targeted inhibitor screening by identifying selective antifolate compounds that could lead to effective treatments for lymphatic filariasis, potentially improving the lives of millions affected by this neglected tropical disease.