A novel and rapid method for the purification of killer toxins

FERNÁNDEZ DE ULLIVARRI M; BELLOMIO A

Córdoba

Congreso; LII Reunión Anual Sociedad Argentina de Investigación en Bioquímica y Biología Molecular; 2016

SAIB

p { margin-bottom: 0.25cm; line-height: 120%; }a:link { }Killer toxins (KTs)are fungal antimicrobial proteins of growing interest in food, healthand pharmaceutical areas. Their application requires previouspurification and characterization; however, traditional purificationmethods are extensive and costly. In this study we describe a rapidand economic method for purification of killer toxin KTCf20, relyingon its affinity to sensitive yeast cells. A suspension of thesensitive strainPichiaguilliermondiiCd6 was produced in YPD at 30 °C,180 rpm for 48 h. Cells were further washed and autoclaved to obtaina dead cell suspension (PDCS) of OD60040. Different interactionconditions between PDCS and a cell-free supernatant (CFS) containingKTCf20 were studied: PDCS:CFS ratio, adsorption time and NaClconcentration, desorption time and temperature. Purification ofKTCf20 was performed from 30 ml of CFS and 100 ml of PDCS. Purifiedwas concentrated by ultrafiltration (3 kDa) and analyzed by SDS-PAGE.Optimal adsorption conditions were: PDCS:CFS 3:1; 10 min; 20 °C.Optimal desorption conditions were: NaCl 1 M; 5 min, 20 °C.Purification was completed in 3.5 h with yield 11.6%. Only one bandwas visualized in SDS-PAGE gel, corresponding to KTCf20. This novelpurification method proved to be faster and less costly thantraditional ones. However, it should be studied on other KTs in orderto validate its effectiveness.