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使用包装床固定金属亲属性染色学对胺标记增强绿色光蛋白 (His-EGFP) 净化的系统优化:多阶段实验设计方法

Yen-Hsun Chou¹, Nguyen The Duc Hanh², Chanin Panjapornpon³

¹Department of Chemical Engineering, Graduate School of Biochemical Engineering, Ming Chi University of Technology, New Taipei City 243303, Taiwan; Department of Chemical Engineering and Materials Science, Yuan Ze University, Zhongli District, Taoyuan City 320315, Taiwan.

International journal of biological macromolecules

|September 4, 2025

概括

这项研究优化了固定金属亲和色谱 (IMAC) 来净化大肠杆菌的His标记增强绿色光蛋白 (His-EGFP). 开发的过程显著提高了His-EGFP的产量和纯度,证明了生物处理的可扩展性.

科学领域:

生物技术
蛋白质净化
生物处理工程

背景情况:

大肠杆菌的重组蛋白质生产对于各种应用至关重要.
有效净化His标记的蛋白质,如His-EGFP对于下游使用至关重要.
固定金属亲和色谱 (IMAC) 是His标签净化的主要方法.

研究的目的:

从重组大肠杆菌中开发和优化His-EGFP的可扩展的IMAC净化过程.
通过实验设计 (DOE) 确定吸附和分离的最佳条件.
评估净化策略的效率,产量和可扩展性.

主要方法:

在大肠杆菌中大规模发酵His-EGFP.
使用超声波破坏细胞.
通过包装床IMAC使用Ni2+充电吸附剂进行净化.
使用一次性一个因子 (OFAT) 和实验设计 (DOE),包括中央复合设计 (CCD),优化吸附和分离.

主要成果:

确定最佳吸附条件:pH8.08,流量6.15毫升/分钟,细胞度64.7% (w/v),床高度15.37厘米.
预测的动态结合能力 (DBC) 达到2.15 × 10^6 AU/mL.
在500mM NaCl中使用300mM imidazole进行优化化.
高吸附效率 (86.68%97.94%) 和最终的His-EGFP产量 (85.45%97.21%) 与良好的净化系数 (3.093.20).

结论:

一个强大的和可扩展的IMAC净化策略成功开发了His-EGFP.
优化过程显著提高了净化效率和产量.
这些发现支持这种方法用于大规模的His-EGFP生物处理.

关键词:

素标记增强的绿色光蛋白 (His-EGFP)固定金属亲和色谱 (IMAC)净化优化

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