Independent valine and leucine isotope labeling in Escherichia coli protein overexpression systems
Independent valine and leucine isotope labeling in Escherichia coli protein overexpression systems
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DOI：
10.1007/s10858-013-9786-y
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发表时间：
2013-11-01
影响因子：
2.7
通讯作者：
Konrat, Robert
中科院分区：
文献类型：
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作者：
Lichtenecker, Roman J.;Weinhaeupl, Katharina;Konrat, Robert
将标记的α-酮基异戊酸酯添加到表达蛋白质的宿主生物的生长介质中，已经演变成一种通用的工具，可以同时将特定的同位素结合到Valine-和Leucine-残基中。由此得到的目标蛋白是蛋白质核磁共振分析的极佳探针。然而，由于这些残基的侧链共振出现在一个狭窄的光谱范围内，信号重叠在高分子量核磁共振探针的情况下表现出严重的限制。我们提出了一种通过向培养液提供未标记的α-酮基异己酸酯来消除亮氨酸标记的方案。得到的模型蛋白质的光谱只具有强度增加的valine信号，证实了该方法是使用α-酮酸前体化合物进行独立的valine和leucine标记的第一个例子。
The addition of labeled alpha-ketoisovalerate to the growth medium of a protein-expressing host organism has evolved into a versatile tool to achieve concomitant incorporation of specific isotopes into valine-and leucine-residues. The resulting target proteins represent excellent probes for protein NMR analysis. However, as the side-chain resonances of these residues emerge in a narrow spectral range, signal overlap represents a severe limitation in the case of high-molecular-weight NMR probes. We present a protocol to eliminate leucine labeling by supplying the medium with unlabeled alpha-ketoisocaproate. The resulting spectra of a model protein exclusively feature valine signals of increased intensity, confirming the method to be a first example of independent valine and leucine labeling employing alpha-ketoacid precursor compounds.