A novel and rapid method for the purification of killer toxins

AUGUSTO BELLOMIO; MIGUEL FERNÁNDEZ DE ULLIVARRI

Córdoba

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

SAIB - Sociedad Argentina de Investigación en Bioquímica y Biología Molecular

Killer toxins (KTs) are fungal antimicrobial proteins of growing interest in food, health and pharmaceutical areas. Their application requires previous purification and characterization; however, traditional purification methods are extensive and costly. In this study we describe a rapid and economic method for purification of killer toxin KTCf20, relying on its affinity to sensitive yeast cells.A suspension of the sensitive strain Pichia guilliermondii Cd6 was produced in YPD at 30 °C, 180 rpm for 48 h. Cells were further washed and autoclaved to obtain a dead cell suspension (PDCS) of OD600 40. Different interaction conditions between PDCS and a cell-free supernatant (CFS) containing KTCf20 were studied: PDCS:CFS ratio, adsorption time and NaCl concentration, desorption time and temperature. Purification of KTCf20 was performed from 30 ml of CFS and 100 ml of PDCS. Purified was concentrated by ultrafiltration (3 kDa) and analyzed by SDS-PAGE.Optimal adsorption conditions were: PDCS:CFS ratio 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 band was visualized in SDS-PAGE gel, corresponding to KTCf20.This novel purification method proved to be faster and less costly than traditional ones. However, it should be studied on other KTs in order to validate its effectiveness.