- Multiple display of protein domains by means of a oligomeric scaffold through recombinant fusion and high affinity leucine zippers peptides.

CRAIG, PO; BERGUER, PM; GOLDBAUM FA

Montevideo, Uruguay

Conferencia; 6th International Conference of Biologycal Physics and 5th Southern Cone Biophysics Congress; 2007

The polymeric display of proteins is a strategy that could be used to increase the immunogenicity of antigens and to enhance the strength of interaction of binding domains for their target ligands through an avidity effect.Brucella Abortus Lumazine Synthase (BLS) is a decameric protein composed of a 17 kDa subunit arranged as a dimer of pentamers. In the past, BLS was successfully used as a carrier to enhance the immunogenicity of short peptides fused to its N termini. However, proteins in their native conformation are required to produce an efficient protective antibody response against pathogens and also for binding specific ligands. In this work, we describe the use of BLS as a oligomeric scaffold for the polymeric display of whole proteins. We present two general strategies for coupling proteins to the structure of BLS: (a) recombinant fusion and linkage through a flexible pentapeptide rich in glicine and serine (GSGSG), and (b) non covalent association through high affinity heterodimerizing leucine zipper peptides (LEU1 and LEU2) complementary fused to the structure of BLS and the target protein. Results obtained with a variety of proteins, indicate that is possible to obtain well folded and stable decameric particles by both methods, although the recombinant fusion through the pentapeptide linker usually needs a refolding step when expressed in E. Coli. The immunological use of BLS as a carrier of antigens is illustrated using a double stranded RNA binding domain derived from murine Staufen-1 protein (RBD3). Immunization of mice with the monomeric RBD3 or RBD3-LEU2 produced no significant immunological response toward this antigen. By contrast, immunization with the decameric RBD3-BLS chimera or RBD3-LEU2::LEU1-BLS ensemble produced a strong and specific immunological response against RBD3, breaking the tolerance of mice to this self antigen. These results demonstrate the potential of the BLS carrier approach to deliver antigens to the immune system in a highly ordered array, preserving their natural conformation and enhancing their immunogenicity. Finally, an increase in the interaction strength of RBD3 for a model double stranded RNA ligand could also be observed when this domain was coupled to BLS.