Protein engineering of BLS to generate a multivalent bifunctional platform

SOSA S; KLINKE S; BERGUER PM; CRAIG PO; NADRA A; GOLDBAUM FA; BONOMI HR

Mar del Plata

Congreso; LI Reunión Anual Sociedad Argentina de Investigación en Bioquímica y Biología Molecular; 2015

Sociedad Argentina de Investigación en Bioquímica y Biología Molecular

The Brucella Lumazine Synthase (BLS) is a homodecameric protein formed by a head-to-head 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 decided 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 aim to produce two different BLS fusion proteins, five copies of each one on a different pentamer and therefore to bifunctionalize BLS in a multivalent manner. 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 and possess the introduction of negatively and positively charged aminoacids in the pentameric interface. These mutations disrupt in cis interactions between pentamers and complement in trans. Both mutant variants have been constructed and purified to homogeneity. Structural analyses demonstrate that BLSa and BLSb form pentamers in solution and when incubated together they are able to heterodimerize (BLSab). In addition, we have created fusion fluorescent proteins (YFP and CFP) to BLSa and BLSb and demonstrated that this system allows the building of a multivalent bifunctional BLSab.