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A simple protocol for amino acid type selective isotope labeling in insect cells with improved yields and high reproducibility

Gossert, Alvar and Hinniger, Alexandra and Gutmann, Sascha and Jahnke, Wolfgang and Strauss, Andre and Fernandez, Cesar (2011) A simple protocol for amino acid type selective isotope labeling in insect cells with improved yields and high reproducibility. Journal of Biomolecular NMR.

Abstract

We have developed a simple protocol for amino acid type selective isotope labeling in insect cells. 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. Similar protein amounts as with full media are obtained, without compromising on isotope incorporation ratios, which are consistently ≥ 90% for all amino acids tested in single and dual amino acid labeling experiments.
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.

Item Type: Article
Keywords: Selective labeling, Insect cells, Baculovirus, Kinase, NMR
Date Deposited: 14 Jun 2016 23:45
Last Modified: 14 Jun 2016 23:45
URI: https://oak.novartis.com/id/eprint/4975

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