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针对GFP的纳米体的工程和特征:表达,净化和翻译后修饰分析

Dunchu Weng¹, Lin Yang¹, Yajun Xie¹

¹The Ministry of Education Key Laboratory of Laboratory Medical Diagnostics, The College of Laboratory Medicine, Chongqing Medical University, Chongqing, 400016, China.

Protein expression and purification

|May 23, 2024

概括

这项研究设计了一个GFP纳米体用于研究,诊断和治疗,证明了其在细菌和哺乳动物表达系统中的有效性. 质谱学揭示了影响纳米体功能的翻译后修改.

科学领域:

生物技术是生物技术.
分子生物学分子生物学
免疫学免疫学免疫学

背景情况:

纳米体是用于研究和治疗的多功能抗体碎片.
选择适当的表达系统对于纳米体功能至关重要,因为折叠和翻译后修饰 (PTM) 的潜在差异.
纳米体工程中PTM的策略尚未得到充分探索.

研究的目的:

设计和验证一个针对绿色光蛋白 (GFP) 的纳米体.
为了比较GFP纳米体在 prokaryotic 和 eukaryotic 系统中的表达和功能.
用质谱学研究PTM对纳米体结合亲和力的影响.

主要方法:

工程GFP纳米体与6×His和Fc标签融合.
在大肠杆菌 (原核细胞) 和HEK293T细胞 (真核细胞) 中表达的纳米体.
使用拉下测试,共免疫沉和质谱 (MS) 进行分析.

主要成果:

在大肠杆菌中产生6×His-GFP-nanobody的高产量生产,能够使GFP蛋白沉.
在HEK293T细胞中成功表达Fc-GFP-nanobody,用于体内GFP标记蛋白质的追踪.
在GFP纳米体中识别出不同的PTM,通过MS表达在 prokaryotic 和 eukaryotic 系统中,与结合差异相关.

结论:

设计的GFP纳米体在原生细胞和真核细胞表达系统中都有效.
表达系统之间的PTM差异影响了纳米体结合能力.
这项研究为功能应用提供了对纳米体工程和表达式优化的洞察.

关键词:

细胞蛋白表达系统是什么?在GFP纳米体.一个纳米人体.这些PTM是PTM.Prokaryotic 蛋白质表达系统是什么?

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