Cost-effective method for the preparation of uniformly labeled myristoylated proteins for NMR measurements.
 

Cost-effective method for the preparation of uniformly labeled myristoylated proteins for NMR measurements.

Related Articles Cost-effective method for the preparation of uniformly labeled myristoylated proteins for NMR measurements.

Protein Expr Purif. 2014 Mar 21;

Authors: Kroupa T, Prchal J, Doležal M, Ruml T, Hrabal R

Abstract
Nuclear magnetic resonance (NMR) is a powerful technique for solving protein structures orstudying their interactions. However, it requires molecules labeled with NMR sensitive isotopes like carbon(13)C and nitrogen(15)N. The recombinant expression of labeled proteins is simple to perform but requires quite expensive chemicals. When there is a need for special labeled chemicals, like uniformly (13)C-labeled myristic acid, the price significantly rises. Here we describe a cost-effective method for the recombinant expression ofuniformly labeled myristoylated proteins in Escherichia coli demonstrated on the production of Mason-Pfizer monkeyvirus matrix protein.We used the ability of E. coli to naturally synthetize myristic acid. Whengrown in isotopically labeled medium the myristic acid will be labelled as well. Bacteria wereco-transfected with plasmid carrying gene for yeast N-myristoyltransferase which ensures myristoylation of expressed protein. This process provided1.8 mgof the myristoylated, doubly labeled ((13)C/(15)N)M-PMV matrix proteinfrom 1 l of (15)N/(13)C labeled M9 medium. The price represents approximately 50% cost reduction of conventional method using commercially available [U-(13)C]myristic acid.


PMID: 24662511 [PubMed - as supplied by publisher]



More...