INVESTIGADORES
BIANCO Maria Isabel
congresos y reuniones científicas
Título:
Preliminary studies for the expression and purification of two proteins form Candidatus Liberibacter asiaticus
Autor/es:
SPERAT W; BIANCO MI; TORRES PS; PAGLIALI F; GONZÁLEZ C; GARCÍA L; MOLINA MC; MARANO, MR; IBAÑEZ LI; VOJNOV AA
Lugar:
Buenos Aires
Reunión:
Congreso; Reunión Conjunta de Sociedades de Biociencias; 2017
Resumen:
Huanglongbing (HLB), disease caused by bacteria of the genusCandidatus Liberibacter (CaLib), is having devastating effects onthe citrus industry worldwide. However, despite its economic importance,little is known about the biology and pathogenesis mechanismsof these bacteria. The main hurdle in the study of CaLib is thatonly one specie is culturable in vitro, Liberibacter crescens, which isnot described as pathogen. Of all the HLB-causing species, CandidatusLiberibacter asiaticus (CaLas) is the most widespread andstudied. Several genomes of CaLas have been sequenced.By analyzing both the sequenced genomes and the recent publicationson the subject, we have selected two proteins that couldpotentially be involved in the pathogenesis of this bacterium: CLIBASIA_04040 (4040), an uncharacterized putative protein, and CLIBASIA_04260, the CaLas OmpA homologue (OmpA).We amplified the sequences corresponding to the 4040 protein(without the N-terminal signal peptide) and the N-terminal solubledomain of OmpA. Both open reading frames were cloned into theexpression vector pET28a(+). Then, we overexpressed 4040 andOmpA in Escherichia coli BL21(DE3) and E. coli Rosetta-gami 2, respectively,both with a hexahistidine-coding sequence. Using differentialcentrifugation, we detected that the 4040 protein was localizedin the soluble fraction (cytoplasm or periplasm), while OmpA waslocalized in the insoluble fraction (inclusion bodies). OmpA was solubilizedby heating the insoluble fraction at to 60 °C for 20 minutes.Our next step is to purify these proteins by affinity chromatographyto analyze them in vitro.