title: Engineering a switchable toxin: the potential use of PDZ domains in the expression, targeting and activation of modified saporin variants.
abstract: A critical problem in studying ribosome-inactivating proteins (RIPs) lies in the very limited possibility to produce them in heterologous systems. In fact, their inherent toxicity for the producing organism nearly always prevents their recombinant expression. In this study, we designed, expressed and characterized an engineered form of the RIP saporin (SapVSAV), bearing a C-terminal extra sequence that is recognized and bound by the second PDZ domain from murine PTP-BL protein (PDZ2). The co-expression of SapVSAV and PDZ2 in Escherichia coli BL21 cells greatly enhances the production of the toxin in a soluble form. The increase of production was surprisingly not due to protection from bacterial intoxication, but may arise from a stabilization effect of PDZ2 on the toxin molecule during biosynthesis. We found that once purified, SapVSAV is stable but is not toxic to free ribosomes, while it is fully active against human cancer cells. This strategy of co-expression of a toxin moiety and a soluble PDZ domain may represent a new system to increase the production of recombinant toxic proteins and could allow the selection of new extra sequences to target PDZ domains inside specific mammalian cellular domains.
authors.affiliation: Department of Basic and Applied Biology, University of L'Aquila, Via Vetoio snc., Coppito, 67010 L'Aquila, Italy.
authors.forename: Francesco
authors.initials: F
authors.lastname: Giansanti
authors.forename: Luana
authors.initials: L
authors.lastname: Di Leandro
authors.forename: Ilias
authors.initials: I
authors.lastname: Koutris
authors.forename: Giuseppina
authors.initials: G
authors.lastname: Pitari
authors.forename: Maria Serena
authors.initials: MS
authors.lastname: Fabbrini
authors.forename: Alessio
authors.initials: A
authors.lastname: Lombardi
authors.forename: David J
authors.initials: DJ
authors.lastname: Flavell
authors.forename: Sopsamorn U
authors.initials: SU
authors.lastname: Flavell
authors.forename: Stefano
authors.initials: S
authors.lastname: Gianni
authors.forename: Rodolfo
authors.initials: R
authors.lastname: Ippoliti
mesh_terms: D000818:Animals; D002460:Cell Line; D004926:Escherichia coli / Q000235:genetics; D015870:Gene Expression; D006801:Humans; D054731:PDZ Domains; D015202:Protein Engineering / Q000379:methods*; D011500:Protein Synthesis Inhibitors / Q000302:isolation & purification / Q000378:metabolism*; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D054789:Ribosome Inactivating Proteins, Type 1 / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism*; D000076985:Saporins; D014162:Transfection
chemical_list: D011500:Protein Synthesis Inhibitors; D011994:Recombinant Proteins; D054789:Ribosome Inactivating Proteins, Type 1; D000076985:Saporins