Isotope labeling in insect cells.
Target proteins in contemporary NMR are becoming increasingly complex. The interest lies on membrane proteins, multi-domain proteins, secreted proteins with secondary modifications etc., which can often only be expressed in eukaryotic expression hosts. Although E. coli is the organism of choice for expression of proteins in isotope labeled form for NMR studies, bacterial cells have limitations concerning their ability of producing soluble and well-folded proteins of human origin. Insect cells are eukaryotic cells and therefore evolutionary closer to human cells than bacterial cells. Therefore a larger share of human proteins can be functionally expressed in them, for example multi-domain proteins, protein complexes, membrane proteins and proteins requiring post-translational modifications. In order to study these proteins by NMR, they are ideally prepared in isotope labeled form. In this chapter different strategies for isotope labeling in insect cells are described - uniform and amino acid specific. A general introduction to expression with baculovirus infected insect cells is given followed by a detailed descriptions of labeling approaches. The chapter is concluded with case studies, describing successful application of isotope labeling in insect cells for NMR studies including solid-state experiments, ligand binding studies and protein dynamics.
Novartis Institutes for BioMedical Research, Basel, Switzerland. alvar.gossert@novartis.com
Alvar D
AD
Gossert
Wolfgang
W
Jahnke
D000596:Amino Acids / Q000737:chemistry; D000818:Animals; D016367:Baculoviridae / Q000235:genetics; D006801:Humans; D007553:Isotope Labeling / Q000379:methods*; D009036:Moths / Q000166:cytology*; D019906:Nuclear Magnetic Resonance, Biomolecular / Q000379:methods*; D011487:Protein Conformation; D011506:Proteins / Q000737:chemistry*; D018411:Spodoptera / Q000166:cytology*
D000596:Amino Acids; D011506:Proteins