Cloning and expression of the B chain of volkensin, type 2 ribosome inactivating protein from Adenia volkensii harms: co-folding with the A chain for heterodimer reconstitution.
Type 2 ribosome inactivating proteins (RIPs) include some potent plant toxins, among which ricin from Ricinus communis and abrin from Abrus precatorius seeds, have been known for more than a century. Two other type 2 RIPs belong to this class of proteins, both isolated from plants of the same family (Passifloraceae), modeccin and volkensin, from Adenia digitata and Adenia volkensii roots, respectively. Volkensin is probably the most potent plant toxin known, with an LD50 for rats of 50-60 ng/kg. Here we report the cloning, expression and renaturation of recombinant volkensin B chain. Furthermore, starting from separately expressed A and B chains, a co-association procedure was set-up, leading to in vitro heterodimeric volkensin reconstitution. The recombinant heterodimer was characterized by N-terminal sequence analysis and its hemagglutinating activity assessed. In parallel, we have explored the carbohydrate-binding properties of native volkensin with the aim to correlate toxin-specific properties (i.e., axonal transport along neurons) to lectin's sugar-binding preferences.
Chambery
Angela
A
Dipartimento di Scienze della Vita, Seconda Università di Napoli, Via Vivaldi 43, I-81100 Caserta, Italy. angela.chambery@unina2.it
Severino
Valeria
V
Stirpe
Fiorenzo
F
Parente
Augusto
A
D002846:Chromatography, Affinity; D003001:Cloning, Molecular; D019281:Dimerization; D004591:Electrophoresis, Polyacrylamide Gel; D004797:Enzyme-Linked Immunosorbent Assay; D006023:Glycoproteins / Q000378:metabolism; D006386:Hemagglutination Tests; D006801:Humans; D009699:N-Glycosyl Hydrolases / Q000096:biosynthesis* / Q000737:chemistry; D037121:Plant Lectins / Q000096:biosynthesis* / Q000737:chemistry; D017510:Protein Folding; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry; D054790:Ribosome Inactivating Proteins, Type 2
D006023:Glycoproteins; D037121:Plant Lectins; D011994:Recombinant Proteins; D054790:Ribosome Inactivating Proteins, Type 2; C049134:volkensin; D009699:N-Glycosyl Hydrolases