Bordetella pertussis is re-emerging in several countries with a high vaccine uptake. Analysis of clinical isolates revealed antigenic divergence between vaccine strains and circulating strains with respect to P.69 pertactin. Polymorphisms in P.69 pertactin are mainly limited to regions comprised of amino acid repeats, designated region 1 and region 2. Region 1 flanks the RGD motif involved in adherence. Although antibodies against P.69 pertactin are implicated in protective immunity, little is known about the structure and location of its epitopes. Previously we described the localization of mainly linear epitopes of both human sera and mouse monoclonal antibodies (mAbs). To study the location of conformational epitopes and to investigate the effect of variation in P.69 pertactin on vaccine efficacy, we cloned, expressed, and purified 3 naturally occurring P.69 pertactin variants, 3 mutants in which the variable regions are missing, 3 N-terminal mutants and I C-terminal deletion mutant. Here, we describe the procedure to clone, express, and purify up to 0.1 mg P.69,pertactin and its derivatives per 1 ml Escherichia coli culture. (c) 2005 Elsevier Inc. All rights reserved.