Wuchereria bancrofti dihydrofolate reductase expression and purification methods with initial kinetic studies
Presentation Type
Abstract
Faculty Advisor
Nina Goodey
Access Type
Event
Start Date
25-4-2025 12:00 PM
End Date
25-4-2025 1:00 PM
Description
Lymphatic filariasis affects over 657 million people worldwide, Wuchereria bancrofti (Wb) being responsible for 90% of cases. The parasite relies on dihydrofolate reductase (DHFR) for folate metabolism. Methotrexate (MTX), a known DHFR inhibitor, demonstrated to be a potent inhibitor against different species of the enzyme, suggesting its potential effectiveness against Wb DHFR. MTX’s inhibition of human DHFR underscores the need for more selective compounds. The gene encoding Wb DHFR, engineered with an N-terminal His₆-tag, was expressed in E. coli LOBSTR strain and purified by a two-step affinity chromatography process of MTX-agarose resin followed by nickel-nitriloacetic acid (Ni-NTA) purification. The final 2L expression yielded 12.07 g of unpurified protein pellet. The final preparation yielded ~4 mg of purified protein per liter of culture, reaching a concentration of 210 µM. Enzyme activity was assessed using a microplate reader with NADPH as a cofactor and dihydrofolate (DHF) as a substrate. SDS-PAGE analysis confirmed the purity of Wb DHFR, revealing a dominant band near the expected molecular weight (~20 kDa) and minimal contamination. Inhibition by MTX was observed and IC₅₀ determination is ongoing. Further kinetic studies such as KM, kcat, and KI determinations will characterize enzyme behavior and inform the design of selective inhibitors. Identifying structural differences between Wb DHFR and human DHFR will be impactful for designing selective inhibitors that target the parasite while reducing off-target effects. DHFR is a viable therapeutic target for lymphatic filariasis and lays the foundation for future drug discovery efforts.
Wuchereria bancrofti dihydrofolate reductase expression and purification methods with initial kinetic studies
Lymphatic filariasis affects over 657 million people worldwide, Wuchereria bancrofti (Wb) being responsible for 90% of cases. The parasite relies on dihydrofolate reductase (DHFR) for folate metabolism. Methotrexate (MTX), a known DHFR inhibitor, demonstrated to be a potent inhibitor against different species of the enzyme, suggesting its potential effectiveness against Wb DHFR. MTX’s inhibition of human DHFR underscores the need for more selective compounds. The gene encoding Wb DHFR, engineered with an N-terminal His₆-tag, was expressed in E. coli LOBSTR strain and purified by a two-step affinity chromatography process of MTX-agarose resin followed by nickel-nitriloacetic acid (Ni-NTA) purification. The final 2L expression yielded 12.07 g of unpurified protein pellet. The final preparation yielded ~4 mg of purified protein per liter of culture, reaching a concentration of 210 µM. Enzyme activity was assessed using a microplate reader with NADPH as a cofactor and dihydrofolate (DHF) as a substrate. SDS-PAGE analysis confirmed the purity of Wb DHFR, revealing a dominant band near the expected molecular weight (~20 kDa) and minimal contamination. Inhibition by MTX was observed and IC₅₀ determination is ongoing. Further kinetic studies such as KM, kcat, and KI determinations will characterize enzyme behavior and inform the design of selective inhibitors. Identifying structural differences between Wb DHFR and human DHFR will be impactful for designing selective inhibitors that target the parasite while reducing off-target effects. DHFR is a viable therapeutic target for lymphatic filariasis and lays the foundation for future drug discovery efforts.
Comments
Poster presentation at the 2025 Student Research Symposium.