Identification and Characterization of the Small Heat Shock Protein Hsp20 from Oenococcus oeni SD-2a
Identification and Characterization of the Small Heat Shock Protein Hsp20 from Oenococcus oeni SD-2a
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酒酒球菌 SD-2a 中小热激蛋白 Hsp20 的鉴定和表征
DOI：
10.1007/s00284-020-02168-z
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发表时间：
2020-08
影响因子：
2.6
通讯作者：
Mingtao Fan
中科院分区：
文献类型：
--
作者：
Yiman Qi;Dan Liu;Haopeng Yu;Guoqiang Zhang;Mingtao Fan
酒酒球菌在苹果酸-乳酸发酵过程中，可以在恶劣的葡萄酒条件下发挥作用。因此，它是探索抗性基因的重要微生物资源。HSP20是一种重要的小分子热休克蛋白。HSP20保守的一致基序“A-x-G-x-L”揭示了其作为小分子热休克蛋白家族成员的作用。克隆了oeni SD-2a的hsp20基因，构建了重组表达载体pTriEx-hsp20。将重组表达载体转化大肠杆菌BL21(DE3)感受态细胞，用异丙基-β-d-硫代半乳糖苷诱导表达HSP20蛋白。成功表达了oeni SD-2a的hsp20基因，并经SDS-PAGE鉴定为20 kDa的融合蛋白。经镍亲和层析得到纯化的Hsp20蛋白。此外，将BL21(DE3)/Hsp20和BL21(DE3)/Ctrl分别在42℃和52℃、pH值2.0-12.0的高温、0.1%(v/v)和0.2%(v/v)的H_2O_2氧化休克以及430和860 mM的氯化钠渗透胁迫下处理，以比较从oeni SD-2a异源表达Hsp20蛋白对其抗逆性的影响。值得注意的是，当面对不同的胁迫条件时，过表达的Hsp20表现出比对照菌株更强的抗性。结果表明，oeni SD-2a hsp20基因的异源表达显著提高了宿主菌对胁迫条件的抗性。
Oenococcus oeni can exert its function in hostile wine conditions during the malolactic fermentation process. Therefore, it is an important microbial resource for exploring resistance genes. Hsp20 is an important small heat shock protein from O. oeni. The conserved consensus motif “A-x-x-x-x-G-x-L” of Hsp20 announced its role as a member of the small heat shock protein family. The hsp20 gene from O. oeni SD-2a was cloned to create the recombinant plasmid pTriEx-Hsp20. The recombinant plasmid was transformed into Escherichia coli BL21(DE3) competent cells, and the Hsp20 protein was induced by isopropyl-β-d-thiogalactoside (IPTG). The hsp20 gene from O. oeni SD-2a was successfully expressed, and a 20-kDa fusion protein was identified by SDS-PAGE. The purified Hsp20 protein was obtained using Ni-affinity chromatography. Additionally, BL21(DE3)/Hsp20 and BL21(DE3)/Ctrl were treated at high temperatures of 42 and 52 °C, at pH values of 2.0–12.0, under oxidative shock with 0.1% (v/v) and 0.2% (v/v) H2O2, and under an osmotic shock of 430 and 860 mM NaCl to compare the effects of heterologous expression of the Hsp20 protein from O. oeni SD-2a for stress resistance. Notably, Hsp20 overexpression showed enhanced resistance than the control strain did when confronted with different elevated stress conditions. The results demonstrated heterologous expression of the hsp20 gene from O. oeni SD-2a significantly improved the resistance of the host E. coli bacteria against stress conditions.
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影响因子：
1.4
作者：
J. Markwell
通讯作者：
J. Markwell
影响因子：
2.7
作者：
Florian Ronez;N. Desroche;P. Arbault;J. Guzzo
通讯作者：
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影响因子：
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影响因子：
5.8
作者：
Larkin, M. A.;Blackshields, G.;Higgins, D. G.
通讯作者：
Higgins, D. G.
DOI：
--
发表时间：
2001-01
期刊：
--
影响因子：
--
作者：
J. Sambrook;E. Fritsch;T. Maniatis
通讯作者：
J. Sambrook;E. Fritsch;T. Maniatis