Purification of bacterial virulence factor pertactin using high affinity ligands

Abstract

Pertussis, or whooping cough, is a contagious bacterial disease, caused by Bordetella pertussis. It is airborne and causes coughing fits, difficulty in breathing, and even death. Whooping cough is life-threatening but could be prevented by proper immunization. The common format of the acellular vaccine comprises a mixture of pertussis toxin, fimbriae, filamentous hemagglutinin, and the virulence factor pertactin. Purification of pertactin is challenging due to its low abundance in the fermentation broth and typically requires multiple laborious unit operations which results in final product recovery of less than 20 %. This paper explores the possibility of reducing the number of purification steps by using a column functionalized with high-affinity ligands produced by mRNA display technology. A design of experiments (DoE) approach was used to explore the effects of ligand type and process parameters on the purity and recovery of pertactin in the eluate. It was found that elution at pH 3 resulted in complete dissociation of pertactin from the column. On-column washing steps with 0.5 % (v/v) Tween 20 and 1 M NaCl reduced the endotoxin content by a 1000-fold. We present a purification method for pertactin, that results in a purity of 90 % and a recovery of about 100 % in the eluate.