Affordable uniform isotope labeling with (2)H, (13)C and (15)N in insect cells.
For a wide range of proteins of high interest, the major obstacle for NMR studies is the lack of an affordable 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 cytosolic and secreted target proteins.
Novartis Institutes for BioMedical Research, 4002, Basel, Switzerland.
Agnieszka
A
Sitarska
Lukasz
L
Skora
Julia
J
Klopp
Susan
S
Roest
César
C
Fernández
Binesh
B
Shrestha
Alvar D
AD
Gossert
D000596:Amino Acids / Q000737:chemistry; D000818:Animals; D002241:Carbohydrates / Q000737:chemistry; D002247:Carbon Isotopes / Q000737:chemistry; D019476:Insect Proteins / Q000737:chemistry*; D007553:Isotope Labeling / Q000379:methods*; D009587:Nitrogen Isotopes / Q000737:chemistry; D019906:Nuclear Magnetic Resonance, Biomolecular / Q000379:methods; D015203:Reproducibility of Results; D018411:Spodoptera / Q000166:cytology / Q000378:metabolism*
D000596:Amino Acids; D002241:Carbohydrates; D002247:Carbon Isotopes; D019476:Insect Proteins; D009587:Nitrogen Isotopes