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

• We present simple way how to prepare NMR labeled myristoylated proteins.
• The procedure gives labeled myristoylated and non-myristoylated protein in one step.
• Our procedure cost is approximately 50% of the conventional procedure.

Nuclear magnetic resonance (NMR) is a powerful technique for solving protein structures or studying their interactions. However, it requires molecules labeled with NMR sensitive isotopes like carbon 13C and nitrogen 15N. 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 of uniformly labeled myristoylated proteins in Escherichia coli demonstrated on the production of Mason-Pfizer monkey virus matrix protein. We used the ability of E. coli to naturally synthetize myristic acid. When grown in isotopically labeled medium the myristic acid will be labelled as well. Bacteria were co-transfected with plasmid carrying gene for yeast N-myristoyltransferase which ensures myristoylation of expressed protein. This process provided 1.8 mg of the myristoylated, doubly labeled (13C/15N)M-PMV matrix protein from 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.