A simple protocol for amino acid type selective isotope labeling in insect cells with improved yields and high reproducibility
- 655 Downloads
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.
- CRC (1977) CRC handbook of chemistry and physics. CRC Press Inc., ClevelandGoogle Scholar
- Das J, Chen P, Norris D, Padmanabha R, Lin J, Moquin RV, Shen Z, Cook LS, Doweyko AM, Pitt S, Pang S, Shen DR, Fang Q, de Fex HF, McIntyre KW, Shuster DJ, Gillooly KM, Behnia K, Schieven GL, Wityak J, Barrish JC (2006) 2-aminothiazole as a novel kinase inhibitor template. Structure-activity relationship studies toward the discovery of N-(2-chloro-6-methylphenyl)-2-[[6-[4-(2-hydroxyethyl)-1-piperazinyl)]-2-methyl-4-pyrimidinyl]amino)]-1,3-thiazole-5-carboxamide (dasatinib, BMS-354825) as a potent pan-Src kinase inhibitor. J Med Chem 49:6819–6832CrossRefGoogle Scholar
- O’Reilly DR, Miller LK, Luckow VA (1994) Baculovirus expression vectors: a laboratory manual. Oxford University Press, OxfordGoogle Scholar
- Vajpai N, Strauss A, Fendrich G, Cowan-Jacob SW, Manley PW, Grzesiek S, Jahnke W (2008a) Solution conformations and dynamics of ABL kinase-inhibitor complexes determined by NMR substantiate the different binding modes of imatinib/nilotinib and dasatinib. J Biol Chem 283:18292–18302CrossRefGoogle Scholar