Isotope Labeling in Insect Cells

Abstract

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.

Calmodulin disrupts plasma membrane localization of farnesylated KRAS4b by sequestering its lipid moiety

“…13 C-methyl labeling of KRAS4b was performed in ESF 921 Delta Series media (TMILVA) supplemented with Ala (400 mg/liter). 13 C-methyl-labeled Thr (75 mg/liter), Met (100 mg/liter), Ile (150 mg/liter), Leu (100 mg/liter), and Val (50 mg/liter) were added 16 hours after infection based on a protocol adapted from Gossert et al (74,75). NMR titrations (E,E)-farnesol, (Z,Z)-farnesol, and (Z,Z)-FCME were solubilized in DMSO to 100 mM, appropriately diluted in NMR buffer, and added directly to separate NMR samples to a final concentration of 2.5% DMSO.…”

Production of Artificially Doubly Glycosylated, 15N Labeled Prion Protein for NMR Studies Using a pH-Scanning Volatile Buffer System

“…The base structure for this image is PDB 1XYX, which is an NMR structure of the C-terminus of mouse PrP C . 10…”

Isotope Labeling of Proteins in Insect Cells

“…For isotope labeling, in principle, all medium components including lipids and vitamins would need to be replaced with labeled ones, rendering the media extremely expensive. However, many substances are only present in trace amounts and can be safely ignored for isotope-labeling purposes (Gossert & Jahnke, 2012). However, amino acids, sugars, fetal bovine serum (FBS), and pluronic are present in high amounts.…”