IN VIVO Protein Expression | Eurisotop

IN VIVO Protein Expression

Isotope-enriched protein is typically produced by over-expressing the protein in genetically engineered E. coliBL21 (DE3) cells that are cultured in an isotopic medium. Protein induction is started when the cells reach the mid-log phase of growth and is generally stopped after several hours. The cells are cracked open and protein collected and purified. The isotopic growth medium usually contains either 13C6 glucose or 13CD7 glucose as the sole carbon source and 15N enriched ammonium chloride as the sole nitrogen source. Protein yields may be increased by using medium containing amino acids.

Selective amino acid type labeling can often be achieved by adding labeled amino acids directly to the culture medium prior to induction. Eukaryotic expression systems are often relied upon when bacterial systems fail to produce the target protein in a properly folded form. The most common eukaryotic expression system for labeling protein is the baculovirus expression vector system (BEVS) employing insect larvae cells. Yeast and mammalian cells are also used for protein expression. For insect and mammalian media, selective and uniform labeling are possible.

Regardless of the expression system, proteins greater than ~30 KDa generally require deuteration to simplify spectra and reduce line-broadening associated with 1H dipolar coupling.

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