Biomolecular NMR

In vivo Protein Expression

Top Ten Tips for Producing 13C, 15N Protein in Abundance (Application Note 15)

Deborah A. Berthold, et al.
Departments of Chemistry and Biochemistry
University of Illinois at Urbana-Champaign


  • 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)
 

 

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.

 

Frequently Asked Questions 

 

Frequently Asked Questions 

How do I know which minimal media reagents to use to make a protein of a specific uniform labeling pattern?

What algal strain is used to create BioExpress® 1000?

Agmenelum quadriplicatum

What algal strain is used to create Celtone® Powder and Celtone® Complete media?

Chlorella vulgaris

What algal strain is used to create the Amino Acid Mixes product?

Agmenelum quadriplicatum

What algal strain is the Whole Algal Cells product?

Agmenelum quadriplicatum

Kevin Millis, PhD

Kevin Millis, PhD

Senior Scientist, Application Development Manager

Kevin Millis, PhD, is the Senior Scientist and Market Development Manager for all NMR and Mass Spectrometry product lines. Kevin is responsible for Technical Services both internally and externally for all CIL customers as well as being responsible for the application and market development for the CIL products.

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