Rational design and characterization of a novel multivalent multifunctional protein scaffold based on Brucella Lumazine Synthase (BLS) (Poster)

SEBASTIÁN KLINKE; ALEJANDRO NADRA; HERNÁN R. BONOMI; SANTIAGO SOSA; ANDRÉS H. ROSSI; JIMENA RINALDI; FERNANDO A. GOLDBAUM; PAULA BERGUER

Tucumán

Congreso; XLV Reunion Anual SAB 2016 and IX IberoAmerican Congress of Biophysics and III LAFeBS; 2016

Sociedad Argentina de Biofísica (SAB)

The Brucella Lumazine Synthase (BLS) is a homodecameric protein formed by the dimerization of homopentamers. We have previously demonstrated its high quaternary stability and immunogenicity, allowing it to be used as antigen carrier. In this work we aimed to mutate the homodimer interface in order to interrupt the association between homopentamers and simultaneously promote the association between mutant heteropentamers. In this way, we will be able to produce a BLS with different fusion proteins on each side of the scaffold. The mutations have been rationally designed based on its crystallographic structure using the bioinformatic softwares FoldX and Pymol. The BLS mutants have been named as BLSa and BLSb. Structural analyses demonstrate that BLSa and BLSb form pentamers in solution and when incubated together they are able to form a dimer of heteropentamers (BLSab). Biophysical properties of the complex (such as thermal stability and dissociation conditions), do not differ from the wild type BLS. In addition, studies in mice demonstrated that the heteropentamer complex immunogenicity is not different from wild type BLS. However, both pentamers are less immunogenic than BLS. As a concept proof, we have functionalized BLSab with a sialic acid binding domain on one side of the protein and a fluorophore on the other side, in order to label mammalian cells in vitro.