Application Note 26
Uniform Isotopic Labeling of Eukaryotic Proteins in Methyltropic Yeast for High-Resolution NMR Studies – Extension to Membrane Proteins
Ying Fan, Lichi Shi, Vladimir Ladizhansky, and Leonid S. Brown
Departments of Physics and Biophysics Interdepartmental Group
University of Guelph, 50 Stone Road East, Guelph, Ontario, N1G 2W1 Canada
Recent developments in multidimensional solid-state and solution NMR allow structural studies of new challenging protein targets, such as polytopic membrane proteins.1-3 Especially attractive are medically relevant families of eukaryotic channels, transporters,and receptors, such as GPCRs.1,4-6 Unfortunately, uniform isotope labeling of any eukaryotic membrane protein for structural studies by high-resolution NMR can be a daunting task.7-10 While the mammallian and insect cell cultures provide native-like membrane environments and allow specific labeling of certain amino acids, relatively low protein yields can lead to extremely high costs for uniformly labeled samples.10,11 This is aggravated by the difficulty of protein deuteration, which is often required for structural studies of mid-size and large membrane proteins.10 The most common alternative, bacterial expression systems such as E. coli, ensure cost-effective uniform labeling and deuteration, but often fail to produce the native fold of eukaryotic membrane proteins and lack the capability for proper post-translational modifications.1,6,8,9 The third possibility, expression in eukaryotic microbes such as methylotrophic yeast P. pastoris, has not been fully explored for isotopic labeling of membrane proteins. While the uniform isotopic labeling and deuteration of soluble secreted proteins in P. pastoris is well-established12-16 and many eucaryotic membrane proteins have been expressed in yeast for crystallization, no high-resolution NMR spectra suitable for structural studies have been reported for yeast-expressed eukaryotic membrane proteins. In this application note, we report on the successful extension and modification of the cost-effective uniform double (13C, 15N) labeling protocol, previously employed for soluble secreted proteins,16 to a eukaryotic seven-transmembrane helical protein.17
The P. pastoris expression system is very attractive for isotopic labeling of eukaryotic proteins in general, as it combines native-like cellular environment and post-translational modifications with the ease of handling and cost-effectiveness similar to those of bacterial systems.10,14 Importantly, P. pastoris can be grown in fermenters, which can dramatically increase yields of isotopically labeled proteins compared to shake flasks.13 The availability of many different strains and vectors allows optimization of selection of best transformants, cellular targeting, and protein tagging for specific proteins.
The double (13C, 15N) uniform isotope labeling of soluble secreted proteins in P. pastoris relies on a very straightforward protocol, in which the carbon source at the preinduction growth phase (e.g. 13C6-glucose) is replaced by 13C-methanol in the postinduction phase.16 Concentrations and timing of addition of isotope sources have been optimized to ensure the cost-effectiveness and completeness of labeling.14 In view of high-yield functional expression of many nonlabeled mammalian membrane proteins, for example, aquaporins and GPCRs,18-20 in P. pastoris, it is logical to adapt the existing isotope-labeling protocols for these attractive targets.
Read more by downloading the application note.