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Expression, purification and enzymatic characterization of Brugia malayi dihydrofolate reductase
- Source :
- Protein Expression and Purification. 128:81-85
- Publication Year :
- 2016
- Publisher :
- Elsevier BV, 2016.
-
Abstract
- Brugia malayi (B. malayi) is one of the three causative agents of lymphatic filariasis, a neglected parasitic disease. Current literature suggests that dihydrofolate reductase is a potential drug target for the elimination of B. malayi. Here we report the recombinant expression and purification of a ∼20 kDa B. malayi dihydrofolate reductase (BmDHFR). A His6-tagged construct was expressed in E. coli and purified by affinity chromatography to yield active and homogeneous enzyme for steady-state kinetic characterization and inhibition studies. The catalytic activity kcat was found to be 1.4 ± 0.1 s(-1), the Michaelis Menten constant KM for dihydrofolate 14.7 ± 3.6 μM, and the equilibrium dissociation constant KD for NADPH 25 ± 24 nM. For BmDHFR, IC50 values for a six DHFR inhibitors were determined to be 3.1 ± 0.2 nM for methotrexate, 32 ± 22 μM for trimethoprim, 109 ± 34 μM for pyrimethamine, 154 ± 46 μM for 2,4-diaminoquinazoline, 771 ± 44 μM for cycloguanil, and >20,000 μM for 2,4-diaminopyrimidine. Our findings suggest that antifolate compounds can serve as inhibitors of BmDHFR.
- Subjects :
- 0301 basic medicine
Cycloguanil
Gene Expression
Catalysis
Brugia malayi
03 medical and health sciences
chemistry.chemical_compound
parasitic diseases
Dihydrofolate reductase
medicine
Animals
Enzyme kinetics
chemistry.chemical_classification
biology
Chemistry
030111 toxicology
Helminth Proteins
biology.organism_classification
Molecular biology
Recombinant Proteins
Dissociation constant
Tetrahydrofolate Dehydrogenase
030104 developmental biology
Pyrimethamine
Enzyme
Biochemistry
Antifolate
biology.protein
Biotechnology
medicine.drug
Subjects
Details
- ISSN :
- 10465928
- Volume :
- 128
- Database :
- OpenAIRE
- Journal :
- Protein Expression and Purification
- Accession number :
- edsair.doi.dedup.....37ad5e3d43d3c8e1fda94bcf503eb81b