首页|结核分枝杆菌分子伴侣Acr2蛋白的原核表达及其功能分析

结核分枝杆菌分子伴侣Acr2蛋白的原核表达及其功能分析

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目的 在E.coli中表达结核分枝杆菌(Mycobacterium tuberculosis,Mtb)分子伴侣Acr2蛋白,并分析其功能.方法 将重组质粒pET-28a-Acr2转化感受态E.coli BL21(DE3),IPTG诱导表达His-Acr2蛋白,经Ni-NTA层析及Superdex™ 200 10/300 GL凝胶过滤层析纯化获得Acr2蛋白.将Acr2蛋白经热变性(100 ℃温浴15 min)及化学变性(8mol/L尿素,37 ℃温浴4 h)后进行自发再折叠和再组装的复性处理,采用圆二色性光谱法和非变性SDS-PAGE检测变性及复性后Acr2蛋白的二级结构.通过底物结合试验检测Acr2蛋白在体外的分子伴侣功能.结果 纯化Acr2蛋白的相对分子质量约为232 000,纯度达90%以上,浓度约为2mg/mL.Acr2蛋白经变性-复性处理后可恢复其天然二级结构,且在48 ℃条件下可与变性的苹果酸激酶(malate dehydrogenase,MDH)形成稳定复合物.结论 Acr2蛋白经变性-复性处理后可恢复其天然分子构象,且具有体外分子伴侣活性.本研究为制备具有天然活性的结核杆菌蛋白抗原提供了新策略.
Prokaryotic expression and functional analysis of molecular chaperone Acr2 protein of Mycobacterium tuberculosis
Objective To express the molecular chaperone Acr2 protein of Mycobacterium tuberculosis(Mtb)in E.coli and analyze the function.Methods The recombinant plasmid pET-28a-Acr2 was transformed into competent E.coli BL21(DE3),and induced by 1PTG.The expressed His-Acr2 protein was purified by Ni-NTA chromatography and Superdex™200 10/300 GL gel filtration chromatography to obtain Acr2 protein.The Acr2 protein was refolded by spontaneous refolding and reassembly after thermal denaturation(100 ℃ for 15 min)and chemical denaturation(8 mol/L urea,37 ℃ for 4 h).The secondary structure of Acr2 protein before and after denaturation-renaturation was detected by circular dichroism spectroscopy and non-denaturing SDS-PAGE,and the molecular chaperone function of Acr2 protein in vitro was detected by substrate binding assay.Results The purified Acr2 protein had the relative molecular mass of about 232 000,the purity of over 90%,and the concentration of about 2 mg/mL,which recovered its natural secondary structure after denaturation-renaturation,and formed stable complexes with the denatured malate dehydrogenase(MDH)at 48 ℃.Conclusion The Acr2 protein can restore its natural molecular conformation with molecular chaperone activity in vitro after denaturation-renaturation treatment,providing a new strategy for the preparation of Mtb protein antigen with natural activity.

Mycobacterium tuberculosis(Mtb)Small heat shock protein(sHSP)Acr2 proteinDenaturationRenatur-ationMolecular chaperone

张宗欣、赵纪元、孙红宾

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郑州轻工业大学食品与生物工程学院,河南郑州 450002

结核分枝杆菌 小分子热休克蛋白 Acr2蛋白 变性 复性 分子伴侣

河南省自然科学基金面上项目

212300410415

2024

中国生物制品学杂志
中华预防医学会,长春生物制品研究所有限责任公司

中国生物制品学杂志

CSTPCD
影响因子:0.417
ISSN:1004-5503
年,卷(期):2024.37(1)
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