In vivo Protein Expression

 

The overexpression of protein using genetically engineered prokaryotic or eukaryotic cells remains the most efficient way to produce isotope-enriched protein suitable for NMR analysis. Among prokaryotic expression systems, the use of the E. coli BL21(DE3) strain is by far the most popular and cost efficient. For some proteins, such as membrane proteins, overexpression in E. coli results in the recombinant protein being deposited into inclusion bodies in nonsoluble forms, which then requires refolding of the protein using detergents or lipids to gain a biochemically active form. To improve the probability of obtaining properly folded protein without the need for refolding, eukaryotic expression systems are used because these cell types contain more complex molecule machinery (e.g., chaperones) to aid in the folding proteins during expression. The most popular eukaryotic expression systems employ either yeast, insect and mammalian cells. Uniform and most types of selective labeling are possible in all cell types.

Regardless of the method, proteins or complexes greater than ~25 kDa in size typically require deuterium enrichment in order to simplify spectra and reduce the deleterious effects of line-broadening associated with ¹H dipolar coupling. Therefore, minimal media used to express such proteins must be formulated using D₂O. For the investigations of large proteins or complexes, uniformly deuterated glucose as the carbon source is required.

 

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