生物学杂志

生物学杂志 ›› 2022, Vol. 39 ›› Issue (1): 17-.doi: 10. 3969/ j. issn. 2095-1736. 2022. 01. 017

• 研究报告 • 上一篇    下一篇

PelA-Expansin 融合蛋白原核重组表达研究

  

  1. 盐城师范学院 盐城市蛋白工程技术研究中心, 盐城 224051
  • 出版日期:2022-02-18 发布日期:2022-02-15
  • 通讯作者: 赵庆新,博士,教授,研究方向为基因蛋白工程, E-mail:515097815@qq.com
  • 作者简介:魏玲玲,硕士在读,研究方向为微生物学,E-mail:18888109113@163.com
  • 基金资助:
    国家自然科学基金项目(41773103);江苏省盐土生物资源研究重点实验室开放基金项目(JKLBS2017012);2016 年江苏省大学生创新创业项目

Prokaryotic expression of PelA-Expansin fusion protein

  1. Yancheng Protein Engineering Technology Research Center,Yancheng Normal University,Yancheng 224051, China
  • Online:2022-02-18 Published:2022-02-15

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摘要：

扩张蛋白(Expansin)具有削弱细胞壁多糖之间氢键,增强多糖降解酶降解多糖的效率。在食品领域、生物能源领域和生物制药领域,具有广泛的应用价值。自然状态下,Expansin 蛋白在植物、真菌、细菌等生物中的表达量较少,目前,枯草芽孢杆菌Expansin 重组表达研究受到广泛关注。以来源于构巢曲霉的果胶酸裂解酶(PelA)为融合标签,构建高效表达融合蛋白PelA-Expansin 的工程菌newexpansin / pelA-pET-28a(+) / BL21(DE3),在OD600 为0. 4、0. 01 mmol/ L IPTG、15 ℃和150 r/ min 下,表达24 h,融合蛋白产量约为3. 0 mg/ mL。建立融合蛋白一次性Histag亲和纯化工艺,工艺中上样、清洗和洗脱缓冲液中咪唑浓度分别为30、 60 和 500 mmol/ L,目标蛋白纯度约为95%,得率约为75%;融合蛋白协同纤维素酶、木聚糖酶和果胶酸水解酶酶降解多糖的效率分别为300%、150%和125%;为扩张蛋白Expansin 在食品、能源、医药及农业等领域的应用奠定了基础。



关键词: 扩张蛋白基因, 果胶酸裂解酶, 融合表达, 分离纯化, 活性分析

Abstract: Expansin can weaken the hydrogen bond between cell wall polysaccharides and enhance the efficiency of polysaccharide degradation by polysaccharide degrading enzyme. It has a wide range of application value in the field of food, bioenergy and biopharmaceutical.In the natural state, the expression of expansin protein in plants, fungi, bacteria and other organisms is less. At present, the research on the recombinant expression of expansin in Bacillus subtilis has been widely concerned. In this project, the expression project strain (newexpansin/ pelA-pET-28a(+) / BL21(DE3)) was constructed with the pectate lyase A (pelA gene from Aspergillus nidulans) as the fusion tag. At OD600 =0. 4, 0. 01 mmol/ L IPTG, 15 ℃ and 150 r/ min for 24 h, the fusion protein production was about 3. 0 mg/ mL. A one-step his-tag affinity purification process was established for the fusion protein and the concentration of imidazole in the sample loading, cleaning and elution buffer were 30, 60 and 500 mmol/ L, the purity of the target protein was about 95%, and the yield was about 75%. The degradation efficiencies of the fusion protein with cellulase, pectinase and xylanase were 300%,150% and 125%, respectively. This project laid the foundation for the application of expansin in the fields of food, energy, medicine and agriculture.

Key words: expansin, pectate lyase A, fusion expression, isolation and purification, activity analysis

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