title: A simple protocol for amino acid type selective isotope labeling in insect cells with improved yields and high reproducibility.
abstract: 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 [(15)N, (1)H]- and [(13)C, (1)H]-HSQC spectra.
authors.affiliation: Novartis Institutes for BioMedical Research, Basel, Switzerland. alvar.gossert@novartis.com
authors.forename: Alvar D
authors.initials: AD
authors.lastname: Gossert
authors.forename: Alexandra
authors.initials: A
authors.lastname: Hinniger
authors.forename: Sascha
authors.initials: S
authors.lastname: Gutmann
authors.forename: Wolfgang
authors.initials: W
authors.lastname: Jahnke
authors.forename: André
authors.initials: A
authors.lastname: Strauss
authors.forename: César
authors.initials: C
authors.lastname: Fernández
mesh_terms: D000596:Amino Acids / Q000032:analysis / Q000737:chemistry / Q000378:metabolism; D000818:Animals; D016367:Baculoviridae / Q000737:chemistry; D002478:Cells, Cultured; D006801:Humans; D007313:Insecta; D007553:Isotope Labeling / Q000379:methods*; D019906:Nuclear Magnetic Resonance, Biomolecular / Q000379:methods; D051057:Proto-Oncogene Proteins c-akt / Q000032:analysis* / Q000737:chemistry*
chemical_list: D000596:Amino Acids; C494921:AKT2 protein, human; D051057:Proto-Oncogene Proteins c-akt