Independent valine and leucine isotope labeling in Escherichia coli protein overexpression systems

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.