Expression, Purification and Functional Characterization of a Recombinant Refolded var2csa DBL3-X

PABLO FERNANDEZ; NIKOLA VIEBIG; SEBASTIEN DECHAVANNE; JURG GYSIN; ARTUR SCHERF; BENOIT GAMAIN

Boston, MA

Congreso; 3rd Protein Engineering Summit PEGS 2007; 2007

Malaria during pregnancy causes serious disease that is associated with placental sequestration of Plasmodium falciparum infected erythrocytes that adhere to chondroitin sulphate A (CSA). The principal CSA binding ligand associated with sequestration in the placenta is the var2csa P. falciparum erythrocyte membrane protein 1 (PfEMP1). The var2csa gene encodes for six Duffy-binding-like (DBL) domains. Among them, the DBL2-X, DBL3- X and DBL6-e specifically bind to CSA. The development of methods to produce functionally active recombinant proteins represents an important preliminary step towards pregnancy associated malaria vaccine development. A DBL3-X recombinant protein was produced using an Escherichia coli expression system. As the protein was expressed in an insoluble form, a refolding strategy based on the immobilization on a solid support was developed. To obtain a highly purified refolded recombinant protein, a gel filtration chromatography step was used. The refolded DBL3-X was then biochemically and biophysically characterised using dynamic light scattering and circular dichroism. The presence of free thiols was also analyzed. Binding assays to placental cryosections and purified CSA under static and physiologically relevant flow conditions were used to functionally characterize recombinant DBL3-X. Antisera against the recombinant protein raised in rats are also currently evaluated for their capacity to cross-react with different CSA binding parasites and inhibit their adhesion to CSA.