in vivo Protein Expression
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 use of the E. coli BL21(DE3) strain is by far the most popular and cost efficient. For some proteins, such as membrane proteins, in vivo overexpression in E. coli results in the recombinant protein being deposited into inclusion bodies in no soluble 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 in vivo expression. The most popular eukaryotic in vivo expression systems employ either yeast, insect and mammalian cells. Uniform and most types of selective labelling 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 in vivo must be formulated using D2O. For the investigations of large proteins or complexes, uniformly deuterated Glucose as the carbon source is required.
Product Information and Resources
- Top Ten Tips for Producing 13C, 15N Protein in Abundance (Application Note 15)
- Cell-Free Protein Synthesis with 2H/15N/13C-Labeled Amino Acids in H2O for the Production of Perdeuterated Proteins with 1H in the Exchangeable Positions (Application Note 36)
- Uniform Isotope Labeling of Eukaryotic Proteins in Methylotrophic Yeast for High-Resolution NMR Studies – Extension to Membrane Proteins (Application Note 26)
- Isotope Labeling of Alanine Methyl Groups on a Deuterated Background for NMR Studies of High-Molecular-Weight Proteins (Application Note 25)
- Insect Cell Media
- Efficient Uniform Isotope Labeling of Proteins Expressed in Baculovirus-Infected Insect Cells using BioExpress® 2000 (Application Note 14)
- Specific Isotopic Labelling of Methyl Groups Has Extended the Molecular Weight for NMR Studies of Protein Structure and Dynamics (Application Note 16)
- Expression Optimization Using BioExpress® 2000 Media (Application Note 20)
- [2,3-13C,]-Labelled Aromatic Residues as a Means to Improving Signal Intensities and Kick-Starting the Assignments (Application Note 22)
- Pichia pastoris as a Eukaryotic Protein Isotope-Labelling System
- Alanine Probes of Supra-Molecular Structure and Dynamics
- Optimization of BioExpress® Supplementation of M9 Cultures (Application Note 12)
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