Affordable uniform isotope labeling with 2H, 13C and 15N in insect cells.

A simple and affordable protocol was developed to produce uni-form labeled proteins in insect cells. Incorporation levels of 80% can be achieved for 15N and 13C and yields are comparable to expression in full media. For 2H,15N and 2H,13C,15N labeling, incorporation is slightly lower with 75% and 73%, respectively, and yields are typically two-fold reduced. The media were optimized for simplicity, reproducibility, isotope incorporation and cost. They consist of only five components, which are all commercially available at less than 8% of the costs than labeling media from vendors. High isotope incorporation and low cost are achieved by using labeled algal amino acid extracts and exploiting well-known biochemical pathways. The approach was applied to several cytosolic and secreted target proteins