Single-step purification of myristoylated and nonmyristoylated recoverin and substrate dependence of myristoylation level.
Recoverin is cotranslationally modified by the covalent linkage of a myristoyl group to its N terminus. It is a member of a family of Ca(2+)-myristoyl switch proteins. Recombinant myristoylated revoverin is currently produced by the cotransformation of bacteria with recoverin and an enzyme that allows N-myristoylation and by supplementing the culture medium with myristic acid. A large variation in the myristoylation level of recoverin and in the amount of myristic acid supplied to the culture medium can be found in the literature. Moreover, although it is known to strongly affect bacterial growth, the amount of ethanol used to solubilize myristic acid is only scarcely mentioned. To improve our understanding of the parameters responsible for recombinant recoverin myristoylation, the effects of myristic acid and ethanol on recoverin myristoylation and expression levels have been systematically studied. In addition, a single-step purification procedure to produce purified myristoylated and nonmyristoylated recombinant recoverin has also been devised. Finally, sodium myristate has been used as an efficient alternative substrate to achieve high myristoylation and expression levels of recoverin. Given that a large number of proteins are myristoylated, these procedures could be applied to several other proteins in addition to recoverin.
Desmeules
Philippe
P
Unité de Recherche en Ophtalmologie, Centre de Recherche du CHUL, Faculté de Médecine, Université Laval, 2705 Blvd. Laurier, Ste-Foy, Que., Canada G1V 4G2.
Penney
Sara-Edith
SE
Salesse
Christian
C
D000818:Animals; D002417:Cattle; D002847:Chromatography, Agarose; D004591:Electrophoresis, Polyacrylamide Gel; D000431:Ethanol; D009226:Myristates / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D051603:Recoverin / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism*
D009226:Myristates; D011994:Recombinant Proteins; D051603:Recoverin; D000431:Ethanol