A simple protocol for amino acid type selective isotope labeling in insect cells with improved yields and high reproducibility
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An easy to use and robust approach for amino acid type selective isotope labeling in insect cells is presented. It relies on inexpensive commercial media and can be implemented in laboratories without sophisticated infrastructure. In contrast to previous protocols, where either high protein amounts or high incorporation ratios were obtained, here we achieve both at the same time. By supplementing media with a well considered amount of yeast extract, similar protein amounts as with full media are obtained, without compromising on isotope incorporation. In single and dual amino acid labeling experiments incorporation ratios are consistently ≥90% for all amino acids tested. This enables NMR studies of eukaryotic proteins and their interactions even for proteins with low expression levels. We show applications with human kinases, where protein–ligand interactions are characterized by 2D [15N, 1H]- and [13C, 1H]-HSQC spectra.
KeywordsIsotope labeling Amino acid selective Insect cells Baculovirus Kinase NMR
We thank Sylvie Antz and Sébastien Rieffel for maintaining cell stocks, Sihame Haddad and Arun Bhat for helpful feedback on the experimental protocol and René Knecht for the analysis of the amino acid content of yeast extract.
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