EXPRESSIONAND PURIFICATION OFANTIMICROBIAL SNAKIN-2 PEPTIDE IN BACTERIALCELLS

GONZALEZ DE URRETA M,; NAGEL ARIEL; VANESA NAHIRÑAK,; ALMASIA, NATALIA INÉS; CECILIA VAZQUEZ ROVERE

Mendoza

Congreso; XLVIII Reunión Anual de la SAIB; 2012

Sociedad Argentina de Investigación en Bioquímica y Biología Molecular (SAIB)

Snakin-2 (SN2) is a cysteine-rich antimicrobial peptide isolatedfrom Solanum tuberosum tubers that was found to be active againstfungal and bacterial pathogens and belongs to the snakin/GASAfamily. Members of this family are widely distributed among plantspecies and are involved in several aspects of plant developmentand plant responses to biotic or abiotic stress. Given theirantimicrobial activity, expression in bacteria was only possible for afew members of this family and it resulted in a low production andthe aggregation of the protein in inclusion bodies. To obtainpurified protein for biochemical and functional studies wegenerated different constructs carrying SN2 coding sequence,based on the pDEST15 and pDEST17 vectors, which have a GSTand a 6xHis tag, respectively. After induction of the transcription,transformed BL21-AI E. coli strain showed a reduced growth ratecompared to control suggesting a toxic effect of the recombinantprotein. For both constructs, tagged-SN2 expression was detectedin the insoluble fraction so different buffers were tested to achievetotal soluble tagged protein. Finally, employing a glutathionesepharose resin, purified GST-tagged SN2 was obtained and it iscurrently used to in vitro determine its activity againstphytopathogens.