One-step separation of myristoylated and nonmyristoylated retroviral matrix proteins.
N-terminal myristoylation of retroviral matrix proteins is essential for the targeting of the Gag polyproteins to the plasma membrane. To investigate the effect of the myristoylation on the structure and membrane binding ability of the matrix proteins, it is necessary to prepare their myristoylated forms. We present purification of myristoylated matrix proteins of the mouse mammary tumor virus and murine leukemia virus, two morphogenetically distinct retroviruses. The proteins were expressed in Escherichia coli coexpressing a yeast N-myristoyltransferase. This E. coli expression system yielded a mixture of myristoylated and nonmyristoylated matrix proteins. We established efficient one-step metal affinity purification that enabled to obtain pure myristoylated matrix proteins suitable for structural and functional studies.
Laboratory of NMR Spectroscopy, Institute of Chemical Technology, Technická 5, 16628 Prague, Czech Republic; Department of Biochemistry and Microbiology, Institute of Chemical Technology, Technická 5, 16628 Prague, Czech Republic; Institute of Organic Chemistry and Biochemistry, Academy of Sciences of the Czech Republic, v.v.i., IOCB & Gilead Research Center, Flemingovo nám. 2, 16610 Prague, Czech Republic.
Michal
M
Doležal
Aleš
A
Zábranský
Richard
R
Hrabal
Tomáš
T
Ruml
Iva
I
Pichová
Michaela
M
Rumlová
D000818:Animals; D002846:Chromatography, Affinity; D003001:Cloning, Molecular; D009052:Leukemia Virus, Murine / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D051379:Mice; D019814:Myristic Acid / Q000737:chemistry / Q000378:metabolism*; D019906:Nuclear Magnetic Resonance, Biomolecular; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D012192:Retroviridae Infections / Q000821:virology; D012191:Retroviridae Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism*
D011994:Recombinant Proteins; D012191:Retroviridae Proteins; D019814:Myristic Acid
Matrix protein; Mouse mammary tumor virus; Murine leukemia virus; Myristoylation; N-myristoyltransferase; Retrovirus