Selective isotopic labeling of recombinant proteins using amino acid auxotroph strains.
Selective isotopic labeling of recombinant proteins using amino acid auxotroph strains.
复制标题
使用氨基酸营养缺陷型菌株对重组蛋白进行选择性同位素标记。
DOI：
10.1007/978-1-59745-456-8_13
复制
发表时间：
2007
期刊：
影响因子：
--
通讯作者：
Whittaker,JamesW
中科院分区：
文献类型：
--
作者：
Whittaker,JamesW
用稳定的同位素标记蛋白质对于许多分析和结构技术是很重要的，包括核磁共振光谱和质谱学。非选择性标记，即统一标记蛋白质中的所有氨基酸，可以利用容易获得的野生型表达宿主来完成。然而，标记特定的氨基酸通常有优点，残基选择性标记通常需要使用对氨基酸具有营养缺陷性的表达菌株，以便有效地结合同位素标记。营养缺乏菌的行为可能会因为生物合成途径的调节特性、由于生物合成途径的中断而产生的次生营养需求、以及由于代谢中间产物的积累而对环境因素获得的敏感性而变得复杂。因此，为了优化其作为选择性标记蛋白质的表达宿主的性能，对每个营养缺陷型菌株的表型进行表征是很重要的。在重组蛋白(半乳糖氧化酶)中标记酪氨酸的毕赤酵母芳香营养缺陷型菌株的应用将被用来说明选择性标记方法。
Labeling proteins with stable isotopes is important for many analytical and structural techniques, including NMR spectroscopy and mass spectrometry. Nonselective labeling, which uniformly labels all amino acids in the protein, may be accomplished with readily available wild-type expression hosts. However, there are often advantages to labeling a specific amino acid, and residue-selective labeling generally requires the use of an expression strain that is auxotrophic for the amino acid in order to efficiently incorporate the isotopic label. The behavior of an auxotrophic strain may be complicated by the regulatory properties of the biosynthetic pathway, by secondary nutritional requirements resulting from disruption of a biosynthetic pathway, and from acquired sensitivity to environmental factors resulting from build-up of metabolic intermediates. As a result, it is important to characterize the phenotype of the each auxotrophic strain in order to optimize its performance as an expression host for selective labeling of proteins. The application of aromatic auxotroph strains ofPichia pastoristo labeling tyrosines in a recombinant protein (galactose oxidase) will be used to illustrate selective-labeling methods.