磁珠固定化结核分枝杆菌二氢叶酸还原酶及其表征

Characterization of Mycobacterium tuberculosis dihydrofolate reductase immobilized on magnetic nanoparticles

摘要:

比较Ni2+-NTA磁珠和羧基磁珠固定结核分枝杆菌二氢叶酸还原酶 (Mycobacterium tuberculosis dihydrofolate reductase，MtDHFR)，探索适合小分子配体混合物库筛选的MtDHFR固定化方法。重组表达带6×His标签MtDHFR，纯化后表征酶学性质，比较用Ni2+-NTA磁珠和羧基磁珠固定化时相应固定化容量、保留活性、稳定性及对抑制剂响应。结果表明，Ni2+-NTA磁珠对MtDHFR固定化容量为 (93±12) mg/g磁珠 (n=3)，但酶比活保留不超过32%，Ni2+明显抑制酶活性，EDTA与Ni2+呈协同抑制效应，Fe3+无显著干扰。羧基磁珠活化固定MtDHFR的容量 (8.6±0.6) mg/g磁珠(n=3)，固定化酶比活保留 (87±4)% (n=3)。在含50 mmol/L KCl的100 mmol/L HEPES (pH 7.0)中，游离和固定化MtDHFR在0 ℃保存16 h活性都无显著改变，但在25 ℃保存16 h，游离酶活性下降近60%而羧基磁珠固定化MtDHFR活性下降仅35%。甲氨喋呤对游离MtDHFR和固定化MtDHFR的IC50无显著差异 (P>0.05)。综上，Ni2+-NTA磁珠不适合固定化MtDHFR；羧基磁珠固定化MtDHFR能保留活性、热稳定性及对抑制剂的响应，该固定化方法有望用于快速筛选其配体混合物库。

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.