A simple and cost-effective protocol for high-yield expression of deuterated and selectively isoleucine/leucine/valine methyl protonated proteins in Escherichia coli grown in shaker flasks

IF 1.3 3区生物学 Q3 BIOCHEMISTRY & MOLECULAR BIOLOGY

Journal of Biomolecular NMR Pub Date : 2021-02-04 DOI:10.1007/s10858-021-00357-x

Mengli Cai, Ying Huang, John Lloyd, Robert Craigie, G. Marius Clore

引用次数: 6

Abstract

A simple and cost-effective protocol is presented for expression of perdeuterated, Ile/Leu/Val ¹H/¹³C methyl protonated proteins from 100?ml cultures in M9?++?/D₂O medium induced at high (OD₆₀₀?~?10) cell density in shaker flasks. This protocol, which is an extension of our previous protocols for expression of ²H/¹⁵N/¹³C and ¹H/¹³C labeled proteins, yields comparable quantities of protein from 100?ml cell culture to those obtained using a conventional 1 L culture with M9/D₂O medium, while using three-fold less α-ketoisovaleric (1,2,3,4-¹³C₄; 3,4′,4′,4′-d₄) and α-ketobutyric (¹³C₄; 3,3-d₂) acid precursors.

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一种在摇瓶中培养的大肠杆菌中高效表达氘化和选择性异亮氨酸/亮氨酸/缬氨酸甲基质子化蛋白的简单且经济高效的方案

提出了一种简单且经济高效的方法，用于表达100?M9?++?在摇瓶中以高(OD600 ~ 10)细胞密度诱导的/D2O培养基。该方案是我们之前的2H/15N/13C和1H/13C标记蛋白表达方案的扩展，从100?用M9/D2O培养基培养1 L的细胞，而用3倍的α-酮异戊酸(1,2,3,4- 13c4;3,4 '，4 '，4 ' -d4)和α-酮丁基(13C4;3,3-d2)酸前体。

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来源期刊

Journal of Biomolecular NMR 生物-光谱学

CiteScore

6.00

自引率

3.70%

发文量

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.