关键词: Inclusion body Mechanistic model Process analytical technology (PAT) Protein refolding Tryptophan and tyrosine fluorescence

Mesh : Inclusion Bodies / chemistry metabolism Protein Refolding Spectrometry, Fluorescence / methods Recombinant Proteins / chemistry metabolism Tryptophan / chemistry Escherichia coli / metabolism chemistry Tyrosine / chemistry Fluorescence Protein Folding

来  源:   DOI:10.1007/s00216-024-05249-1   PDF(Pubmed)

Abstract:
Inclusion bodies (IBs) are protein aggregates formed as a result of overexpression of recombinant protein in E. coli. The formation of IBs is a valuable strategy of recombinant protein production despite the need for additional processing steps, i.e., isolation, solubilization and refolding. Industrial process development of protein refolding is a labor-intensive task based largely on empirical approaches rather than knowledge-driven strategies. A prerequisite for knowledge-driven process development is a reliable monitoring strategy. This work explores the potential of intrinsic tryptophan and tyrosine fluorescence for real-time and in situ monitoring of protein refolding. In contrast to commonly established process analytical technology (PAT), this technique showed high sensitivity with reproducible measurements for protein concentrations down to 0.01 g L - 1 . The change of protein conformation during refolding is reflected as a shift in the position of the maxima of the tryptophan and tyrosine fluorescence spectra as well as change in the signal intensity. The shift in the peak position, expressed as average emission wavelength of a spectrum, was correlated to the amount of folding intermediates whereas the intensity integral correlates to the extent of aggregation. These correlations were implemented as an observation function into a mechanistic model. The versatility and transferability of the technique were demonstrated on the refolding of three different proteins with varying structural complexity. The technique was also successfully applied to detect the effect of additives and process mode on the refolding process efficiency. Thus, the methodology presented poses a generic and reliable PAT tool enabling real-time process monitoring of protein refolding.
摘要:
包涵体(IBs)是由于重组蛋白在大肠杆菌中过表达而形成的蛋白聚集体。尽管需要额外的加工步骤,但IBs的形成是重组蛋白生产的有价值的策略。即,隔离,溶解和重折叠。蛋白质重折叠的工业过程开发是一项劳动密集型任务,主要基于经验方法而不是知识驱动的策略。知识驱动的流程开发的先决条件是可靠的监控策略。这项工作探索了内在色氨酸和酪氨酸荧光用于实时和原位监测蛋白质重折叠的潜力。与通常建立的过程分析技术(PAT)相比,该技术显示了高灵敏度,可重复测量蛋白质浓度低至0.01gL-1。重折叠过程中蛋白质构象的变化反映为色氨酸和酪氨酸荧光光谱最大值位置的变化以及信号强度的变化。峰值位置的移动,表示为光谱的平均发射波长,与折叠中间体的量相关,而强度积分与聚集程度相关。这些相关性被实现为机械模型中的观察函数。在具有不同结构复杂性的三种不同蛋白质的重折叠上证明了该技术的多功能性和可转移性。该技术还成功地应用于检测添加剂和工艺模式对重折叠工艺效率的影响。因此,提出的方法提出了一个通用和可靠的PAT工具,使蛋白质重折叠的实时过程监测。
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