Characterization of Mycobacterium tuberculosis dihydrofolate reductase immobilized on magnetic nanoparticles.

Wei Zhou; Jinpeng LU; Yaping LI; Linyu Yang; Xiaolei HU; Fei Liao; Xiaolan Yang

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Characterization of Mycobacterium tuberculosis dihydrofolate reductase immobilized on magnetic nanoparticles.

Author: Wei ZHOU ¹ ; Jinpeng LU ¹ ; Yaping LI ¹ ; Linyu YANG ¹ ; Xiaolei HU ¹ ; Fei LIAO ² ; Xiaolan YANG ¹
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1. Key Laboratory of Medical Laboratory Diagnostics of the Ministry of Education of China, College of Laboratory Medicine, Chongqing Medical University, Chongqing 400016, China.
2. School of Pharmacy and Bioengineering, Chongqing University of Technology, Chongqing 400054, China.
Publication Type:Journal Article
Keywords: dihydrofolate reductase; immobilization; ligand mixture; magnetic particles; screening
MeSH: Enzyme Stability; Enzymes, Immobilized; Hydrogen-Ion Concentration; Kinetics; Ligands; Magnetite Nanoparticles; Mycobacterium tuberculosis; Temperature; Tetrahydrofolate Dehydrogenase
From: Chinese Journal of Biotechnology 2019;35(3):513-521
CountryChina
Language:Chinese
Abstract: To explore the immobilization of target proteins for screening libraries of ligand mixtures, magnetic submicron particles (MSP) functionalized with Ni²⁺-NTA and carboxyl were compared for the immobilization of Mycobacterium tuberculosis dihydrofolate reductase (MtDHFR). MtDHFR fused with 6×His was expressed, purified and characterized for kinetics. MtDHFR was immobilized on Ni²⁺-NTA-functionalized MSP directly and carboxyl-functionalized MSP upon activation. The immobilization capacity, residual activity, thermostability and affinities for putative inhibitors were characterized. MtDHFR immobilized on Ni²⁺-NTA-functionalized MSP retained about 32% activity of the free one with the immobilization capacity of (93±12) mg/g of MSP (n=3). Ni²⁺ and EDTA synergistically inhibited MtDHFR activity, while Fe³⁺ had no obvious interference. MtDHFR immobilized on carboxyl-functionalized MSP retained (87±4)% activity of the free one with the immobilization capacity of (8.6±0.6) mg/g MSP (n=3). In 100 mmol/L HEPES (pH 7.0) containing 50 mmol/L KCl, there was no significant loss of the activities of the free and immobilized MtDHFR after storage at 0 °C for 16 h, but nearly 60% and 35% loss of their activities after storage at 25 °C for 16 h, respectively. The inhibition effects of methotrexate on the immobilized and free MtDHFR were consistent (P>0.05). The immobilization of MtDHFR on carboxyl-functionalized MSP was thus favorable for higher retained activity and better thermostability, with promise for rapid screening of its ligand mixtures.