IIBBA   05544
INSTITUTO DE INVESTIGACIONES BIOQUIMICAS DE BUENOS AIRES
Unidad Ejecutora - UE
congresos y reuniones científicas
Título:
Rational design and characterization of a novel multivalent bifunctional protein scaffold based on Brucella Lumazine Synthase (BLS)
Autor/es:
RINALDI, J.; BERGUER, P.; SOSA SANTIAGO; ROSSI, A.; GOLBAUM, F.A.; KLINKE S; NADRA A; BONOMI, H.R.
Lugar:
San Miguel de Tucumán
Reunión:
Congreso; XLV Reunión Anual de la Sociedad Argentina de Biofísica; 2016
Institución organizadora:
SAB
Resumen:
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 an antigen carrier. In this work we aimed to engineer the homodimer interface in order to disrupt the association between homopentamers and simultaneously promote the associationbetween mutant heteropentamers. In this way, we desigend a system to produce BLS molecules with di erent functions on each side of the decameric sca old. The mutations have been rationally designedbased on its crystallographic structure using the bioinformatic softwares FoldX and Pymol. The BLS mutants have been named as BLSDR and BLSKE. Structural analyzes demonstrate that BLSKE and BLSDRbehave as pentamers in solution and when incubated together they form a stable dimer of heteropentamers (BLSDRKE). Biophysical properties of the complex (such as thermal stability and dissociation), areidentical to the wild type BLS. In addition, studies in mice demonstrate that the heteropentamer complex immunogenicity is similar to the wild type BLS. Moreover, both pentamers alone are less immunogenicthan the BLS decamer. As a proof of concept, we have functionalized BLSDRKE with sialic acid binding activity on one pentamer and uororescence on the other, to label mammalian cells in vitro, successfullyrevealed by ow cytometry. The strategy presented in this work provides a novel, simple and easy to manipulate protein engineered platform to obtain multiple polarized functions in one molecule withpossible applications in biotechnology, biomedicine and nanotechnology.