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

Publication date: Available online 21 March 2014
Source:Protein Expression and Purification</br>
Author(s): Tomáš Kroupa , Jan Prchal , Michal Doležal , Tomáš Ruml , Richard Hrabal</br>
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 carbon13C and nitrogen15N. 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 13C-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 (13C/15N)M-PMV matrix proteinfrom 1 l of 15N/13C labeled M9 medium. The price represents approximately 50% cost reduction of conventional method using commercially available [U-13C]myristic acid.
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