Expression and purification of antimicrobial snakin-2 peptide in bacterial cells

GONZALEZ DE URRETA, M; NAGEL, A; NAHIRÑAK, V; ALMASIA, NI; VAZQUEZ- ROVERE, C

Mendoza

Congreso; XLVIII Reunión anual de la Sociedad Argentina de Investigaciones en Bioquímica y Biología Molecular; 2012

SAIB

Snakin-2 (SN2) is a cysteine-rich antimicrobial peptide isolated from Solanum tuberosum tubers that was found to be active against fungal and bacterial pathogens and belongs to the snakin/GASA family. Members of this family are widely distributed among plant species and are involved in several aspects of plant development and plant responses to biotic or abiotic stress. Given their antimicrobial activity, expression in bacteria was only possible for a few members of this family and it resulted in a low production and the aggregation of the protein in inclusion bodies. To obtain purified protein for biochemical and functional studies we generated different constructs carrying SN2 coding sequence, based on the pDEST15 and pDEST17 vectors, which have a GST and a 6xHis tag, respectively. After induction of the transcription, transformed BL21-AI E. coli strain showed a reduced growth rate compared to control suggesting a toxic effect of the recombinant protein. For both constructs, tagged-SN2 expression was detected in the insoluble fraction so different buffers were tested to achieve total soluble tagged protein. Finally, employing a glutathione sepharose resin, purified GST-tagged SN2 was obtained and it is currently used to in vitro determine its activity against phytopathogens.