IN VIVO Protein Expression
Producing isotope-enriched protein directly in living cells
The in vivo 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 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 non-soluble forms, which then requires refolding using detergents or lipids to regain a biochemically active form. To improve the probability of obtaining properly folded protein without the need for refolding, eukaryotic expression systems are used, since these cell types contain more complex molecular machinery (e.g., chaperones) to aid in protein folding during expression. The most popular eukaryotic expression systems employ yeast, insect, or 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 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 in vivo must be formulated using deuterium oxide (D2O). For the investigation of large proteins or complexes, uniformly deuterated glucose as the carbon source is required.
Going Further with Eukaryotic Expression
While prokaryotic systems remain the fastest and most economical route to labeled protein, eukaryotic systems — yeast, insect, and mammalian cells — offer a higher probability of properly folded, biochemically active protein, particularly for complex or membrane-associated targets. Our Eukaryotic Cell Growth Expression page details our dedicated growth media for each system, including BioExpress® 2000 for insect cells and BioExpress® 6000 — the only commercially available labeled mammalian media for producing NMR-grade labeled protein.
Related Products
Minimal Media Reagents
Core components for formulating D2O-based minimal media for in vivo isotope labeling.
Alpha-Keto Acids
Precursors used for selective amino acid-type labeling during protein expression.
Methyl-Labeling Kits
Ready-to-use kits for targeted methyl-group labeling in large protein complexes.
Minimal & Rich Bacterial Media
Formulated media for E. coli-based expression of isotope-labeled protein.
Yeast Media and Reagents
Media formulations for producing properly folded, labeled protein in yeast cells.
Insect Cell Media
BioExpress® 2000 rich growth media for baculovirus-based insect cell expression.
Mammalian Cell Media
BioExpress® 6000, the only commercially available labeled mammalian media for NMR-grade protein.
