磁珠固定化结核分枝杆菌二氢叶酸还原酶及其表征
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国家自然科学基金 (Nos. 31570862,81773625) 资助。


Characterization of Mycobacterium tuberculosis dihydrofolate reductase immobilized on magnetic nanoparticles
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National Natural Science Foundation of China (Nos. 31570862, 81773625).

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    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.

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周伟,卢进鹏,李亚平,杨林玉,胡小蕾,廖飞,杨晓兰. 磁珠固定化结核分枝杆菌二氢叶酸还原酶及其表征[J]. 生物工程学报, 2019, 35(3): 513-521

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  • 收稿日期:2018-07-31
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  • 在线发布日期: 2019-03-22
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