Publication date: Available online 21 March 2014 Source:Protein Expression and Purification
Author(s): Tomáš Kroupa , Jan Prchal , Michal Doležal , Tomáš Ruml , Richard Hrabal
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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A cost-effective look at proteins - with a little help from robotics - Cordis News
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A cost-effective look at proteins - with a little help from robotics
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Until now, a number of methods have been used to study static protein structures, including X-ray crystallography and nuclear magnetic resonance. However, these methods are of no use for proteins that are on the move; analytical methods, computer ...
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Cost-effective method for the preparation of uniformly labeled myristoylated proteins for NMR measurements
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