Ni2+诱导的His标记的重组蛋白的选择性沉缩短了净化时间,同时保持了高产量
Md Din Islam1, M Monirul Islam2, Yutaka Kuroda3
1Department of Biotechnology and Life Science, Faculty of Engineering, Tokyo University of Agriculture and Technology, 2-24-16 Nakamachi, Koganei-shi, Tokyo 184-8588, Japan.
Journal of biotechnology
|January 15, 2025
概括
一种新方法使用自由离子 (Ni2+) 来通过从大肠杆菌溶解物沉来快速净化His标记蛋白质. 这种方法提供了高特异性和与传统色谱相比的产量,保留了蛋白质的功能.
科学领域:
- 生物化学 生物化学
- 蛋白质净化 蛋白质净化
- 分子生物学分子生物学
背景情况:
- -NTA亲和色谱是His标记蛋白质净化的标准.
- 传统的方法耗时,劳动密集型,并且可以降低蛋白质产量.
研究的目的:
- 开发一种更简单,更快,更高效的方法来净化标记His的蛋白质.
- 利用自由Ni2+从细胞溶解物中对His标记的蛋白质进行分量沉.
主要方法:
- 使用直接从大肠杆菌溶解物中获得的自由Ni2+ (1mM最终度) 的His标记蛋白质的分量沉.
- 使用10%的酸溶解沉蛋白质.
- 通过逆相HPLC进行进一步净化.
主要成果:
- 成功净化了三种不同的His6标记的重组蛋白质.
- 通过SDS-PAGE证实了Ni2+降水的几乎完全特异性.
- 每200毫升培养液产生3.58.0毫克的产量,与Ni-NTA染色学相比或更高.
- 纯化蛋白质保留了原生折叠和结合活性 (ELISA,BLI).
结论:
- 2+沉方法是一种简单,快速和有效的替代His标记蛋白质净化方法.
- 这种方法适用于小型和大型应用.
- 这种技术保持了蛋白质的完整性和功能.
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