Display and release of the Plasmodium falciparum circumsporozoite protein using the autotransporter MisL of Salmonella enterica Patricia Ruiz-Olvera, a,b Fernando Ruiz-P erez, a Nicol as Villegas Sepulveda, c Araceli Santiago-Machuca, a Rogelio Maldonado-Rodr  ıguez, b Guadalupe Garcia-Elorriaga, a and C esar Gonz alez-Bonilla a, * a Unidad de Investigaci onM edica en Inmunolog  ıa e Infectolog  ıa, Hospital de Infectolog  ıa ‘‘Dr. Daniel M endez Hern andez,’’ Centro M edico ‘‘La Raza,’’ IMSS, Mexico, D.F. b Escuela Nacional de Ciencias Biol ogicas del IPN, M exico, D.F. c Biomedicina Molecular, Centro de Investigaci on y de Estudios Avanzados del IPN, M exico, D.F. Received 17 February 2003, revised 1 May 2003 Abstract The Salmonellaenterica MisL(proteinofmembraneinsertionandsecretion)isanautotransporterwithhighhomology toAIDA-I(adhesininvolvedindiffuseadherence)ofenteropathogenic Escherichia coli.Consideringthatithasbeenre- portedthattheMisL b translocatordomainisabletodisplayheterologouspassengerpeptidestothebacterialsurface,we developed a system to display proteins and release them to the external environment by means of proteolytic cleavage. Plasmidswereconstructedencoding8or53repeatsoftheNANP(Asp–Ala–Asp–Pro)tetrapeptide,whichisthemainB cellepitopeofthe Plasmodiumfalciparum circumsporozoiticprotein(CSP),fusedtothetheMisL b-domainandincluding the recognition cleavage sequence from the E. coli OmpT surface protease. E. coli XL-10Gold and BL21(DE3) (OmpT positive and negative, respectively) and Salmonella enterica serovar Typhimurium SL3261 (Aro A  ) were transformed with the plasmids and, both expression and localization of the fusion proteins were assessed by Western blot, indirect immunofluorescence, and flow cytometry, using a monoclonal antibody against (NANP) 3 . Higher expression of the (NANP) 8 and(NANP) 53 fusionproteinswasdemonstratedonthebacterialsurfaceoftheOmpTnegative E.coli strains and the (NANP) 53 in the culture supernatant of E. coli XL-10Gold indicating a protease mediated cleavage. The flow cytometryanalysissuggested71and98%cleavageefficiencyforthe(NANP) 8 and(NANP) 53 ,respectively,in E.coli XL- 10Gold.Similarresultswereobtainedin S. enterica serovar Typhimurium SL3261, suggesting the involvement of other proteasesrelatedtoOmpT.TheseresultsdemonstratethatMisLmaybeusedfortheautodisplayandreleaseofpassenger proteinsinattenuated Salmonella or E.coli strains,whichmayhaveseveralapplicationsinvaccinedesign. Ó 2003PublishedbyElsevierScience(USA). Keywords: Autodisplay; Salmonella enterica; MisL; Plasmodium falciparum; Live vector vaccines; Circumsporozoite protein Plasmid 50 (2003) 12–27 www.elsevier.com/locate/yplas * Corresponding author. Fax: (55)5583-0626. E-mail address: bonilla@sni.conacyt.mx (C. Gonz alez-Bonilla). 0147-619X/03/$ - see front matter Ó 2003 Published by Elsevier Science (USA). doi:10.1016/S0147-619X(03)00047-7