探索以丙酮酸为核磁共振唯一碳源的细菌培养基的生化景观。

IF 1.3 3区 生物学 Q3 BIOCHEMISTRY & MOLECULAR BIOLOGY
Çağdaş Dağ, Kerem Kahraman
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引用次数: 0

摘要

利用大肠杆菌生产重组蛋白是结构生物学的基石,特别是核磁共振(NMR)光谱研究。了解大肠杆菌在不同碳源下的代谢行为对于优化同位素标记策略至关重要,而同位素标记策略对于核磁共振测定蛋白质结构至关重要。最近的进展,如混合丙酮酸标记,已经改善了大型蛋白质的主链共振分配,使得选择性同位素标记策略比以往任何时候都更重要。在这项研究中,我们的目的是研究大肠杆菌在丙酮酸作为唯一碳源生长时的代谢适应性,丙酮酸是一种常用的条件,用于实现核磁共振光谱的选择性标记。利用基于核磁共振的代谢组学,我们跟踪了整个培养过程中的关键代谢变化,以更好地了解丙酮酸代谢如何影响蛋白质产生和同位素标记。我们的研究结果表明,在IPTG诱导之前,丙酮酸迅速耗尽,而乙酸和乳酸则由于溢出代谢而积累。这些副产物在诱导后仍然存在,表明丙酮酸被转移到废物途径中,这限制了其在同位素结合中的有效利用。这种代谢效率低下对依赖丙酮酸作为核磁共振研究碳源的同位素标记方案提出了挑战。我们的研究结果强调需要微调丙酮酸补充以提高代谢效率和同位素标记,使本研究与优化涉及蛋白质结构测定的核磁共振研究方案直接相关。这些见解为提高核磁共振光谱中同位素标记蛋白质的质量和产量提供了有价值的指导。
本文章由计算机程序翻译,如有差异,请以英文原文为准。
Exploring the biochemical landscape of bacterial medium with pyruvate as the exclusive carbon source for NMR studies.

The use of Escherichia coli for recombinant protein production is a cornerstone in structural biology, particularly for nuclear magnetic resonance (NMR) spectroscopy studies. Understanding the metabolic behavior of E. coli under different carbon sources is critical for optimizing isotope labeling strategies, which are essential for protein structure determination by NMR. Recent advancements, such as mixed pyruvate labeling, have enabled improved backbone resonance assignment in large proteins, making selective isotopic labeling strategies more important than ever for NMR studies. In this study, we aimed to investigate the metabolic adaptations of E. coli when grown on pyruvate as the sole carbon source, a common condition used to achieve selective labeling for NMR spectroscopy. Using NMR-based metabolomics, we tracked key metabolic shifts throughout the culture process to better understand how pyruvate metabolism affects protein production and isotopic labeling. Our results reveal that pyruvate is rapidly depleted before IPTG induction, while acetate and lactate accumulate due to overflow metabolism. These byproducts persist after induction, indicating that pyruvate is diverted into waste pathways, which limits its efficient use in isotope incorporation. This metabolic inefficiency presents a challenge for isotopic labeling protocols that rely on pyruvate as a carbon source for NMR studies. Our results highlight the need to fine-tune pyruvate supplementation to improve metabolic efficiency and isotopic labeling, making this study directly relevant to optimizing protocols for NMR studies involving protein structure determination. These insights provide valuable guidance for enhancing the quality and yield of isotopically labeled proteins in NMR spectroscopy.

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来源期刊
Journal of Biomolecular NMR
Journal of Biomolecular NMR 生物-光谱学
CiteScore
6.00
自引率
3.70%
发文量
19
审稿时长
6-12 weeks
期刊介绍: The Journal of Biomolecular NMR provides a forum for publishing research on technical developments and innovative applications of nuclear magnetic resonance spectroscopy for the study of structure and dynamic properties of biopolymers in solution, liquid crystals, solids and mixed environments, e.g., attached to membranes. This may include: Three-dimensional structure determination of biological macromolecules (polypeptides/proteins, DNA, RNA, oligosaccharides) by NMR. New NMR techniques for studies of biological macromolecules. Novel approaches to computer-aided automated analysis of multidimensional NMR spectra. Computational methods for the structural interpretation of NMR data, including structure refinement. Comparisons of structures determined by NMR with those obtained by other methods, e.g. by diffraction techniques with protein single crystals. New techniques of sample preparation for NMR experiments (biosynthetic and chemical methods for isotope labeling, preparation of nutrients for biosynthetic isotope labeling, etc.). An NMR characterization of the products must be included.
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