Crowley Erika L, Rafferty Steven P
Environmental and Life Sciences Graduate Program, Trent University, 1600 West Bank Drive, Peterborough, ON, K9J 0G2, Canada.
Department of Chemistry, Trent University, 1600 West Bank Drive, Peterborough, ON, K9J 0G2, Canada.
Protein Expr Purif. 2019 May;157:70-85. doi: 10.1016/j.pep.2019.01.007. Epub 2019 Jan 30.
NMR is an important method in the structural and functional characterization of proteins, but such experiments typically require isotopic labelling because of the low natural abundance of the nuclei of interest. Isotope-labelled protein for NMR experiments is typically obtained from IPTG-inducible bacterial expression systems in a minimal media that contains labelled carbon or nitrogen sources. Optimization of expression conditions is crucial yet challenging; large amounts of labelled protein are desired, yet protein yields are lower in minimal media, while the labelled precursors are expensive. Faced with these challenges there is a growing body of literature that apply innovative methods of induction to optimize the yield of isotope-labelled protein. A promising technique is lactose-driven auto-induction as it mitigates user intervention and can lead to higher protein yields. This review assesses the current advances and limitations surrounding the ability of researchers to isotope label proteins using auto-induction, and it identifies key components for optimization.
核磁共振(NMR)是蛋白质结构和功能表征的重要方法,但由于目标原子核的天然丰度较低,此类实验通常需要进行同位素标记。用于NMR实验的同位素标记蛋白质通常从IPTG诱导的细菌表达系统中获得,该系统位于含有标记碳源或氮源的基本培养基中。表达条件的优化至关重要但具有挑战性;需要大量的标记蛋白质,但在基本培养基中蛋白质产量较低,而标记前体又很昂贵。面对这些挑战,越来越多的文献采用创新的诱导方法来优化同位素标记蛋白质的产量。一种有前景的技术是乳糖驱动的自诱导,因为它减少了用户干预并能提高蛋白质产量。本综述评估了围绕研究人员使用自诱导对蛋白质进行同位素标记的能力的当前进展和局限性,并确定了优化的关键组成部分。