PRODUCTION OF RECOMBINANT SITE-SPECIFIC ACETYLATED PEPTIDES IN Escherichia coli

BUSTOS, D. M.; FRONTINI LÓPEZ, Y. R.; UHART, M.

Mendoza

Congreso; XXXIV Reunión Científica Anual de la Sociedad de Biología de Cuyo; 2016

Sociedad de Biologia de Cuyo

p { margin-bottom: 0.25cm; direction: ltr; color: rgb(0, 0, 10); line-height: 120%; text-align: left; }p.western { font-family: "Calibri",serif; font-size: 11pt; }p.cjk { font-family: "Calibri"; font-size: 11pt; }p.ctl { font-size: 11pt; }a:link { }Productionof peptides with non-natural or modified amino acids requires highexpertise and usually special chemical equipment. Recently,genetically modified microoganisms that express a specific tRNA andaminoacyl-tRNA synthase allow the incorporation of posttranslationally modified amino acids like acetylated lysine (acK) orphosphorylated serines. 14-3-3 regulatory proteins are readers ofpostranslationally modified proteins in the proteome. These proteinsrecognize a phosphoserine or threonine in their targets clients.Interesting, 14-3-3 activity is regulated by acetylation. It has beenshown that lysine 49 acetylation (acK49) blocks the binding of 14-3-3to their target proteins. We aimed at producing a peptide containingacK49 surrounded by amino acids corresponding to the wild typesequence of human 14-3-3 to be used as epitope for antibodyproduction. E.coliBL21 were electroporated with two plasmids which express (1) theaminoacyl-tRNA sintetase specific to AcK and (2) the 14-3-3 peptide(with the codon corresponding to K49 mutated to introduce an acK)fused to His6-taggedhuman histone H3 as carrier protein. Positive selection was performedusing chloramphenicol and kanamycin, corresponding to both plasmidsresistance genes. Once selected the colonies containing bothplasmids, they were cultured in LB medium until OD600=0.5 in the presence of antibiotics. Expression induction was carriedout through addition of 0.5 mM IPTG, 0.2% w/v L-arabinose, 1 mMnicotinamide and 1 mM Nε-acetyl-L-lysineto the culture medium. We obtained a pure and soluble acetylatedpeptide in a concentration near to 1 mg x liter of culture medium.This method allows the production of site-specific modificatedpeptides in bacteria, which serves as a great low-cost tool to studythe effect of postranslational modifications in proteins.