Independent valine and leucine isotope labeling in Escherichia coli protein overexpression systems.
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 sidechain 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.
Roman J
RJ
Lichtenecker
Katharina
K
Weinhäupl
Lukas
L
Reuther
Julia
J
Schörghuber
Walther
W
Schmid
Robert
R
Konrat
D002247:Carbon Isotopes / Q000737:chemistry; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism*; D029968:Escherichia coli Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D015870:Gene Expression; D007553:Isotope Labeling; D007930:Leucine / Q000737:chemistry* / Q000378:metabolism; D019906:Nuclear Magnetic Resonance, Biomolecular; D014633:Valine / Q000737:chemistry* / Q000378:metabolism
D002247:Carbon Isotopes; D029968:Escherichia coli Proteins; D007930:Leucine; D014633:Valine