Alternative use of a flexible linker or high affinity heterodimerizing leucine zippers peptides for protein coupling to a oligomeric scaffold

CRAIG PO; BERGUER PM; GOLDBAUM FA

Ventura, California, USA

Congreso; Gordon Research Conference on Biomolecular Interactions & Methods; 2008

<!-- /* Style Definitions */ p.MsoNormal, li.MsoNormal, div.MsoNormal {mso-style-parent:""; margin:0in; margin-bottom:.0001pt; mso-pagination:widow-orphan; font-size:12.0pt; font-family:"Times New Roman"; mso-fareast-font-family:"Times New Roman"; mso-ansi-language:ES; mso-fareast-language:ES;} @page Section1 {size:8.5in 11.0in; margin:1.0in 1.25in 1.0in 1.25in; mso-header-margin:.5in; mso-footer-margin:.5in; mso-paper-source:0;} div.Section1 {page:Section1;} --> 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 this work, we describe the use of BLS as a oligomeric scaffold for the polymeric display of 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 protein models (9 kDa double stranded RNA binding domain from murine Staufen-1 protein, 18 kDa sialic acid binding domain from Bovine Rotavius VP4 capsid protein, and 26 kDa jellyfish Green Fluorescent protein) 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 and not always succeed. We used CD, fluorescence, and light scattering techniques for the analysis of these constructions and also a original method based on the analysis of thermal unfolding curves as a function of urea concentration. These method dissects the unfolding behavior of the protein domains and allows a detailed analysis of the integrity, stability and independent folding structure of the constituting modules. The immunological use of BLS as a carrier of antigens is illustrated with the RBD3 antigen. 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 demonstrates the potential of the BLS carrier approach to deliver antigens to the immune system in a highly order array, preserving their natural conformation and enhancing their immunogenicity. In addition, 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. In this work we present valuable data for the design, production and characterization of macromolecular assemblies and multidomain proteins for its use in the bionanotechnology field.