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.

Isotope Labeling in Insect Cells

“…Labeled amino acid is added in such an excess to yield a targeted fi nal incorporation ratio. We showed an incorporation ratio of 80% for 15 N-Leu labeling of Abl Kinase with this approach [ 5 ] .…”

“…In this step, attention needs to be paid to the fact, that insect cells don't tolerate large changes in the medium very well. Changing from a commercial medium to a medium prepared in house usually results in lowered protein yields [ 5 ] . Therefore, pre-cultures should be maintained in a very similar medium as the main culture.…”

“…With the published protocols, isotope incorporation of maximally 90-94% can be obtained [ 5,13 ]. Unlabeled components, which are present in the labeling medium as described above, are not the main reason for lowered incorporation.…”

“…Two approaches have been followed: Preparing the media from scratch [ 1 ] or ordering amino acid-free versions of commercial media, i.e. without amino acids and yeast extract [ 2,5,13,18 ] .…”

“…At the same time incorporation ratios are only reduced by 2% compared to the yeast extract-free approach. Knowledge of the amino acid composition of yeast extract enabled adjusting its amount in such a way that no amino acid was present in the medium at more than 5% in unlabeled form [ 5 ] .…”