Isotopic labeling of proteins in non-bacterial expression systems

As therapeutic proteins gain importance alongside traditional small molecule drugs, there is increasing interest in using NMR methods to examine their structure, dynamics, and stability/aggregation in solution.

Modern heteronuclear NMR of proteins relies on isotopically-labeled samples containing NMR active nuclei in the peptide backbone, sidechains, or both.

Although isotopic-labeling of recombinant protein is typically carried out in E. Coli expression systems, many biotherapeutic proteins must be expressed in eukaryotic systems to insure proper folding and/or post-translational modifications.   In practice, this means overexpression in either yeast, insect or mammalian cells.

Increased interest in attaining labeled protein samples for analysis by NMR is leading to better commercial availability of isotopically-labeled expression media and improved vectors for overexpression in non-bacterial systems.

Comprehensive reviews of state-of-the-art protocols and procedures for expression of isotopically-labeled proteins in non-standard systems are available here: yeast, insect cells, and mammalian cells.

 

 

Important to use deuterated buffers in small molecule NMR

One way to make your life massively easier if you are doing NMR of small molecules, especially at low concentrations (sub-1mM), is to simply work out what buffer you’d like to use and then order all of the components in deuterated form ahead of time.

For example, if you would like to study your molecule in a buffer like HEPES with 5% DMSO, you can order fully-deuterated HEPES and DMSO from companies like CIL and Sigma-ISOTEC.  Although expensive, the time it can save you at the spectrometer and the enhanced quality of the data are likely worthwhile tradeoffs.

You can also go a step further and prepare your buffers in 100% D2O, making water suppression vastly easier and improving the quality of your spectra. These steps work together in a synergistic manner to dramatically improve your data quality when acquiring on small molecules at low concentrations.