INVESTIGADORES
BUSTOS Diego Martin
congresos y reuniones científicas
Título:
PRODUCTION OF RECOMBINANT SITE-SPECIFIC ACETYLATED PEPTIDES IN Escherichia coli
Autor/es:
FRONTINI, YESICA; UHART, MARINA; BUSTOS, DIEGO M.
Lugar:
MEndoza
Reunión:
Congreso; Sociedad de Biología de Cuyo; 2016
Institución organizadora:
Sociedad de Biología de Cuyo
Resumen:
AbstractProduction of peptides with non-natural or modified amino acids requires high expertise and usually specialchemical equipment. Recently, genetically modified microoganisms that express a specific tRNA and aminoacyl-tRNA synthetase allow the incorporation of post-translationally modified amino acids like acetylated lysine (AcK)or phosphorylated serines. 14-3-3 regulatory proteins are readers of postranslationally modified proteins in the pro-teome. These proteins recognize a phosphoserine or threonine in its targets clients. Interestingly, 14-3-3 activity isregulated by acetylation. It has been shown that lysine 49 acetylation (AcK49) blocks the binding of 14-3-3 to theirtarget proteins. We aimed at producing a peptide containing AcK49 surrounded by amino acids corresponding tothe wild type sequence of human 14-3-3 to be used as epitope for antibody production. E. coli BL21 were elec-troporated with two plasmids which express (1) the aminoacyl-tRNA sinthetase specific to AcK and (2) the 14-3-3peptide (with the codon corresponding to K49 mutated to UAG) fused to His6-tagged human histone H3 as carrierprotein. Positive selection was performed using chloramphenicol and kanamycin, corresponding to both plasmidsresistance genes. Once selected the colonies containing both plasmids, they were cultured in LB medium untilOD600= 0.5 in the presence of antibiotics. Expression induction was carried out through addition of 0.5 mM IPTG,0.2% w/v L-arabinose, 1 mM nicotinamide and 1 mM N-acetyl-L-lysine to the culture medium. We obtained apure and soluble acetylated peptide in a concentration near to 1 mg per liter of culture medium. This method allowsthe production of site-specific modified peptides in bacteria, which serves as a great low-cost tool to study the effectof postranslational modifications in proteins.
