Expression and purification of myristoylated matrix protein of Mason-Pfizer monkey virus for Structural Studies

Jan Prchal^a,b, Petra Junkova^b, Miroslava Strmiskova^a, Jan Lipov^b, Radovan Hynek^b, Tomas Ruml^b and Richard Hrabal^a

^aLaboratory of NMR Spectroscopy and ^bDepartment of Biochemistry and Microbiology, Institute of Chemical Technology, Prague, Technicka 5, 16628 Prague, Czech Republic

Matrix proteins (MA) are the N-terminal domains of structural polyproteins Gag of all retroviruses. They play multiple roles both in the early and late stages of the viral replication cycle. Most of the retroviral matrix proteins are N-terminally myristoylated which is a common posttranslational modification of eukaryotic proteins. The myristoylation is necessary for transport of immature virus particles to the cytoplasmic membrane and for budding of virus particle out of the cell. Unfortunately prokaryotic expression systems, like Escherichia coli, are unable to produce myristoylated proteins. To overcome this problem and yet retain the advantage of strong expression in bacterial cells we co-expressed the matrix protein with yeast N-myristoyl transferase. We managed to prepare a C-terminally His-tagged matrix protein of Mason-Pfizer monkey virus in a large quantity and a high purity using a single-step purification procedure. This protein has been used for NMR and MALDI-TOF studies. The MS analysis proved a high degree of myristoylation of the MA (more than 95%). The comparison of NMR spectra of the matrix protein with and without the myristic acid revealed substantial structural changes of the myristoylated protein.

This research was supported financially by the Czech Science Foundation grant 203/07/0872 and by the Czech Ministry of Education Grants 1M6837805002, MSM 6046137305 and ME 904.