Highly efficient residue-selective labeling with isotope-labeled Ile, Leu, and Val using a new auxotrophic E-coli strain
Highly efficient residue-selective labeling with isotope-labeled Ile, Leu, and Val using a new auxotrophic E-coli strain
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DOI：
10.1007/s10858-016-0042-0
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发表时间：
2016-06-01
影响因子：
2.7
通讯作者：
Kainosho, Masatsune
中科院分区：
文献类型：
--
作者：
Miyanoiri, Yohei;Ishida, Yojiro;Kainosho, Masatsune
我们最近开发了一种实用的方案，用于制备带有立体选择性C-13-甲基标记的亮氨酸和缬氨酸的蛋白质，而不是通常使用的C-13-甲基标记的这些氨基酸的前体，通过E. coli细胞表达。使用该方案，制备具有同位素标记或未标记的Leu和瓦尔残基的任何组合的蛋白质，包括不能通过前体方法制备的一些。然而，使用标记的前体或瓦尔本身的方法，在瓦尔残基的标记效率方面仍有改进的空间。这是由于即使加入大量的瓦尔或其前体也不能充分抑制瓦尔的生物合成。在本研究中，我们通过使用来自大肠杆菌的突变菌株彻底解决了这个问题。大肠杆菌BL 21（DE 3），其中依赖于两种酶（二羟酸脱氢酶和β-异丙基苹果酸脱氢酶）的代谢途径通过缺失分别编码这两种酶的ilvD和leuB基因而完全中止。Delta ilvD E.大肠杆菌突变体终止了从α，β-二羟基异戊酸到α-酮异戊酸的转化，以及从α，β-二羟基-α-甲基戊酸到α-酮-β-甲基戊酸的转化，其分别产生瓦尔和Ile的前体。通过进一步缺失leuB基因，从瓦尔到Leu的转化也完全终止。利用双缺失突变体Δ ilvD Δ leuB E. coli BL 21（DE 3）中，建立了一种高效的、选择性标记Ile、Leu和瓦尔残基的方法。
We recently developed a practical protocol for preparing proteins bearing stereo-selectively C-13-methyl labeled leucines and valines, instead of the commonly used C-13-methyl labeled precursors for these amino acids, by E. coli cellular expression. Using this protocol, proteins with any combinations of isotope-labeled or unlabeled Leu and Val residues were prepared, including some that could not be prepared by the precursor methods. However, there is still room for improvement in the labeling efficiencies for Val residues, using the methods with labeled precursors or Val itself. This is due to the fact that the biosynthesis of Val could not be sufficiently suppressed, even by the addition of large amounts of Val or its precursors. In this study, we completely solved this problem by using a mutant strain derived from E. coli BL21(DE3), in which the metabolic pathways depending on two enzymes, dihydroxy acid dehydratase and beta-isopropylmalate dehydrogenase, are completely aborted by deleting the ilvD and leuB genes, which respectively encode these enzymes. The Delta ilvD E. coli mutant terminates the conversion from alpha,beta-dihydroxyisovalerate to alpha-ketoisovalerate, and the conversion from alpha,beta-dihydroxy-alpha-methylvalerate to alpha-keto-beta-methylvalerate, which produce the preceding precursors for Val and Ile, respectively. By the further deletion of the leuB gene, the conversion from Val to Leu was also fully terminated. Taking advantage of the double-deletion mutant, Delta ilvD Delta leuB E. coli BL21(DE3), an efficient and residue-selective labeling method with various isotope-labeled Ile, Leu, and Val residues was established.