Expression of recombinant Clostridium difficile toxin A using the Bacillus megaterium system SilkeBurger,HelmaTatge,FredHofmann,HaraldGenth,IngoJust,andRalfGerhard * Institute of Toxicology, Hanover Medical School, Hanover, Germany Received19May2003 Abstract Pathogenic Clostridium difficile producestwomajorproteintoxins,toxinAandtoxinB.Weusedthe Bacillus megaterium ex- pressionsystemforexpressionofrecombinanttoxinA.TheconstructforthetoxinAgenewasobtainedbythefollowingcloning strategy:thegenefortoxinAwasgeneratedinthreeparts,eachofthemligatedintoacloningvector.Thethreepartswerese- quentiallyfusedtothecompletegene.TheholotoxingenewasligatedintotheexpressionvectorpWH1520.Thisvectorwasmodified togenerateatoxinwithaC-terminallylocatedHis-tag.Geneexpressioninthe B. megaterium systemresultedinanapproximate 300kDaprotein,whichwasidentifiedbyspecificantibodyastoxinA.Recombinant,His-taggedtoxinAwaspurifiedbyNi 2þ aswell asthyroglobulinaffinitychromatography.CharacterizationoftherecombinanttoxinAshowedidenticalcytotoxicityandinvitro- glucosyltransferaseactivityasthenativetoxinAfrom C. difficile. Ó 2003ElsevierInc.Allrightsreserved. Keywords: Cytotoxicityassay;Endotoxin-freeexpressionsystem;Glucosyltransferase;pWH1520vector Currently, the most common cause of nosocomial diarrhea is the Clostridium difficile-associated diarrhea (CDAD) [1]. Antibiotics predispose patients to coloni- zationwith C. difficile bydisruptingthenormalfloraof the gut. Symptoms of C. difficile infection vary from milddiarrheatosevereformsofintestinalinflammation. The causative agents of the disease are toxin A (308kDa)andtoxinB(270kDa).Kellyandco-workers [2]showedinananimalmodelthatisolatedtoxinAwas able to induce both secretory diarrhea and inflamma- tion.SinceonlytoxinAwasabletocausesecretionand inflammation in an animal model it was designated as theenterotoxin[3].Incontrast,toxinBisaboutthou- sandtimesmorecytotoxicthantoxinAuponcultivated cells and is therefore designated as cytotoxin. Both toxins are intracellular acting glucosyltransferases that transfer a glucose moiety from the cosubstrate UDP- glucosetotargetproteinsoftheRhoGTPasesubfamily Rho, Rac, and Cdc42 [4]. Conventional purification of both toxins is currently performed by ion exchange chromatography, resulting in crude protein fractions highly enriched with toxin A or toxin B. Biologically fullyactivetoxinAisfurtherpurifiedbythyroglobulin affinity purification [5]. Alternatively, Zn 2þ affinity chromatographyhasalsobeendescribed[6].However,it cannot be excluded that these affinity-purified toxin A fractions are contaminated with additional biologically activeclostridialproducts. Tolearnmoreabouttheinflammatoryprocessesin- duced by toxin A it would be of great advantage to possess recombinantly expressed toxin. Recombinant toxin A, free of chaperons or co-purified clostridial products, is a sophisticated tool to study signal trans- duction processes in the pathogenesis of intestinal in- flammation. So far, only toxin fragments, e.g., the receptor binding domain, have been cloned and ex- pressed recombinantly [7]. To our experience the pure repetitivesequenceoftheC-terminallylocatedreceptor binding domain shows weaker competition with the holotoxin in cytotoxicity assays than larger toxin frag- ments.Isolatedfragmentsmaybelesspotentcompared to the identical domain within the holotoxin. Several hintsindicatethatsmallchangesinthetertiarystructure BiochemicalandBiophysicalResearchCommunications307(2003)584–588 www.elsevier.com/locate/ybbrc BBRC * Correspondingauthor.Fax:+49-511-532-2879. E-mail address: gerhard.ralf@mh-hannover.de (R.Gerhard). 0006-291X/03/$-seefrontmatter Ó 2003ElsevierInc.Allrightsreserved. doi:10.1016/S0006-291X(03)01234-8