info@ evotec.com www.evotec.com ISOTOPE-LABELED PROTEINS FOR NMR High-yield production of isotope-labeled protein for NMR Optimised expression protocols for prokaryotic and eukaryotic expression Bespoke labeling strategies to facilitate specific NMR assays State of the art purification and protein analysis Short turn-around times to obtain high-quality, labeled protein at low cost Over ten years experience in isotope-labeling & a track record of > 100 labeled proteins YOU/ WE PROVIDE ATGC WE PROCESS 2 15 Gene sequence N WE DELIVER H C 13 Plasmid E.coli NMR analysis 15 Nominated protein target N 13 C P. pastoris Labeled protein 15 N/13CLeu, Tyr, Phe, Val, Met, … Cell/virus Insect cells info@ evotec.com www.evotec.com Evotec Protein Production Services IN-HOUSE OPTIMISED CULTURE MEDIA AND PROTOCOLS FOR COST-EFFICIENT EXPRESSION OF ISOTOPE LABELED PROTEINS: E. coli P. pastoris Insect cells Ubiquitous labeling 15 Site-speciﬁc labeling* 13 Media properties NH4 as N-source, glucose (E. coli) and methanol (P. pastoris) as C-source, dedicated trace metal and vitamin mixes N, 13 C, 2H C- Val, Leu/ Ile using α-keto -isovalerate/ -butyrate 15 N, 13 C N, 13C-labeling possible, but afﬁliated with high media costs 15 – CASE STUDY 1 15 N- and/or 13C for: Val, Leu, Met, Tyr, Phe, Ile, His, Trp, Thr – taylor-made minimal media – CASE STUDY 2 Expression of a 15N-labeled serine protease in Pichia pastoris was optimised, e.g. expression time / temperature The purified protease was submitted to an NMR fragment screen using 15 N-HSQC Active-site 1H-15N-resonances could be identified by analysing chemical shift perturbations induced by benzamidine binding: Expression of a 15N/13C-Met/Leu-labeled kinase in insect cells Due to the presence of a unique LeuMet dipeptide, the 1H-15N-signal of the gatekeeper Met could be identified using CCLP-HSQC (see below). This facilitated compound binding studies where X-ray crystallography failed NH 105 Highly experienced team and state-of-the-art technology platform for membrane protein production, isotope-labeled protein preparation, high throughput protein purification and structural biology services Mammalian cells 1 105 NH 2 110 110 115 115 H,15N HSQC spectrum 1 ω1 - 15H (ppm) ω1 - 15H (ppm) LEU 120 125 125 130 130 MET 135 135 11 10 9 8 ω2 - 1H (ppm) 7 6 11 10 9 ω2 - 1H (ppm) 8 7 Reagent production services can be accessed as a standalone function or as integral part of wider collaborative programmes Links to key academic groups from Oxford University & Diamond light source, UK H,15N-CCLP-HSQC LEU 120 Direct communication with project scientists and full access to raw data 6