生物学杂志

生物学杂志 ›› 2023, Vol. 40 ›› Issue (2): 27-.doi: 10.3969/j.issn.2095-1736.2023.02.027

• 研究报告 • 上一篇    下一篇

疏水类弹性蛋白多肽的克隆表达及相变特性分析

王珊珊1,4， 刘　阳1， 高　琪1， 王　令1,4， 路宏朝1,2， 张　涛1,3,4   

  1. 1. 陕西理工大学 生物科学与工程学院， 汉中 723001； 2. 陕西省资源生物重点实验室， 汉中 723001；
    3. 陕南秦巴山区生物资源综合开发协同创新中心， 汉中 723001； 4. 陕西理工大学 秦巴生物资源与生态
    环境省部共建国家重点实验室（培育）， 汉中 723001
  • 出版日期:2023-04-18 发布日期:2023-04-18
  • 通讯作者: 张涛，博士，教授，研究方向为生物化学与分子生物学，E-mail: zhangtao780823@snut.edu.cn
  • 作者简介:王珊珊，博士，讲师，研究方向为多肽与蛋白质工程，E-mail: wss@snut.edu.cn
  • 基金资助:
    陕西省教育厅专项科研计划项目（20JK0574）； 陕西省教育厅重点科学研究计划项目（20JY006）

Cloning, expression and analysis of phase transition characteristics of hydrophobic elastin-like polypeptides

WANG Shanshan1,4, LIU Yang1, GAO Qi1, WANG Ling1,4, LU Hongzhao1,2, ZHANG Tao1,3,4   

  1. 1. School of Biological Science and Engineering, Shaanxi University of Technology, Hanzhong 723001, China;
    2. Shaanxi Province Key Laboratory of Bio-resources, Hanzhong 723001, China; 3. QinLing-Bashan Mountains
    Bioresources Comprehensive Development C. I. C., Hanzhong 723001, China; 4. Qinba State Key Laboratory of
    Biological Resources and Ecological Environment, Shaanxi University of Technology, Hanzhong 723001, China
  • Online:2023-04-18 Published:2023-04-18

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摘要： 类弹性蛋白多肽(elastin-like polypeptides，ELPs)作为新型温敏生物材料，在药物递送、组织工程和蛋白质分离纯化领域具有广阔的应用前景，精确控制快速生物合成具有特定相变特性的ELPs至关重要。利用质粒重建递归定向连接技术，成功构建以缬氨酸为客座氨基酸的重组质粒pET28-ELP25、pET28-ELP50和pET28-ELP100，分别转化至Escherichia coli BL21(DE3)中，经自诱导培养基的培养，SDS-PAGE结果表明，ELPs在大肠杆菌体系中实现了胞内可溶过量表达，表达量约占胞内全细胞蛋白的30%以上。利用可逆相变循环法(inverse transition cycling，ITC)进行分离纯化，仅需3轮ITC获得电泳纯ELP25、ELP50和ELP100。热比浊法测定相变温度，分析显示ELPs的相变温度与多肽浓度、离子强度和多肽分子量均呈负相关，为进一步重组克隆表达特定相变特性的温敏多肽生物材料奠定理论基础。

关键词: 类弹性蛋白多肽, 质粒重建递归定向连接, 可逆相变循环, 相变温度

Abstract: Elastin-like polypeptides (ELPs) as novel thermal-sensitive biomaterials have a broad application prospect in drug delivery, tissue engineering and protein isolation and purification. It is essential for precise control and rapid biosynthesis of ELPs with specific phase transition characteristics. Technology of recursive directional ligation by plasmid reconstruction was used to successfully construct recombinant plasmids pET28-ELP25, pET28-ELP50 and pET28-ELP100 as valine as guest amino acid of ELPs. Then, the recombinant plasmids were transformed into Escherichia coli BL21(DE3), respectively. After cultures of recombinant strains under self-induced medium, SDS-PAGE results showed that ELPs were successfully soluble over-expressed in E. coli system, and the expression level was over 30% of total cell proteins. ELP25, ELP50 and ELP100 were separated and purified by three rounds of inverse transition cycling (ITC). Thermal turbidimetry was used for measurement of phase transition temperature (Tt) of ELPs. Analysis of phase transition characteristics revealed that the Ttof ELPs was negatively correlated with polypeptide concentration, ionic strength and molecular weight of ELPs. The results provide a theoretical basis for further recombinant expression of thermal-sensitive polypeptide biomaterials with required phase transition characteristic.

Key words: elastin-like polypeptides, recursive directional ligation by plasmid reconstruction; inverse transition cycling, phase transition temperature

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