ARTICLE Affordable uniform isotope labeling with 2 H, 13 C and 15 N in insect cells Agnieszka Sitarska 1 • Lukasz Skora 1 • Julia Klopp 1 • Susan Roest 1 • Ce ´sar Ferna ´ndez 1 • Binesh Shrestha 1 • Alvar D. Gossert 1 Received: 16 March 2015 / Accepted: 22 April 2015 Ó Springer Science+Business Media Dordrecht 2015 Abstract For a wide range of proteins of high interest, the major obstacle for NMR studies is the lack of an af- fordable eukaryotic expression system for isotope labeling. Here, a simple and affordable protocol is presented to produce uniform labeled proteins in the most prevalent eukaryotic expression system for structural biology, namely Spodoptera frugiperda insect cells. Incorporation levels of 80 % can be achieved for 15 N and 13 C with yields comparable to expression in full media. For 2 H, 15 N and 2 H, 13 C, 15 N labeling, incorporation is only slightly lower with 75 and 73 %, respectively, and yields are typically twofold reduced. The media were optimized for isotope incorporation, reproducibility, simplicity and cost. High isotope incorporation levels for all labeling patterns are achieved by using labeled algal amino acid extracts and exploiting well-known biochemical pathways. The final formulation consists of just five commercially available components, at costs 12-fold lower than labeling media from vendors. The approach was applied to several cy- tosolic and secreted target proteins. Keywords Eukaryotic cells Á Insect cells Á Protein expression Á Isotope labeling Á Uniform isotope labeling Á NMR Á Deuteration Introduction Current biological questions can often only be studied with proteins produced in eukaryotic expression systems. For structural biology, insect cells—typically Sf9 and Sf21 cells (Vaughn et al. 1977)—have become the eukaryotic expression host of choice. For NMR studies, however, isotope labeling has been limited to amino acid specific labeling (Bru ¨ggert et al. 2003; Strauss et al. 2003; Taka- hashi and Shimada 2009; Gossert et al. 2011; Gossert and Jahnke 2012) and only recently, more affordable protocols for uniform 15 N and 2 H, 15 N labeling have become avail- able (Strauss et al. 2005; Kofuku et al. 2014; Meola et al. 2014). Here we present a robust protocol for uniform la- beling of 2 H, 13 C and 15 N in insect cells, which yields high isotope incorporation levels and at the same time is inex- pensive and simple to use. In order to obtain uniformly isotope labeled proteins in insect cells, all sources of amino acids in the growth medium need to be replaced with labeled ones. Since insect cell metabolism is limited, these sources are primarily pure amino acids added to the medium and yeast extract. Gen- erally, an amino acid-free basal medium is ordered from vendors (in this work amino acid free Sf-900 TM III from Gibco LifeTechnologies, and amino acid free SF-4 from Bioconcept) and the desired amino acids are added. There are several strategies in order to replace unlabeled amino acids. Addition of all amino acids in their pure form with the desired isotope label typically results in prohibitive prices. A slightly less expensive alternative is to purchase ready made labeling media (e.g. Bioexpress-2000, CIL), which however still cost roughly 10,000 and 20,000 USD per liter for 15 N and 15 N, 13 C labeling, respectively (Strauss et al. 2005). Additionally, these media are not available for 2 H, 15 N or 2 H, 13 C, 15 N labeling. Another possibility is to use Electronic supplementary material The online version of this article (doi:10.1007/s10858-015-9935-6) contains supplementary material, which is available to authorized users. & Alvar D. Gossert alvar.gossert@novartis.com 1 Novartis Institutes for BioMedical Research, 4002 Basel, Switzerland 123 J Biomol NMR DOI 10.1007/s10858-015-9935-6