Abstract
The addition of labeled α-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 α-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 α-ketoacid precursor compounds.
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Acknowledgments
This work was supported by grants from the Austrian Science foundation FWF P20549, P22125 and W1221B03.
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Lichtenecker, R.J., Weinhäupl, K., Reuther, L. et al. Independent valine and leucine isotope labeling in Escherichia coli protein overexpression systems. J Biomol NMR 57, 205–209 (2013). https://doi.org/10.1007/s10858-013-9786-y
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DOI: https://doi.org/10.1007/s10858-013-9786-y