Clonagem e expressão da proteína LIPL31 de leptospira interrogans

Abstract

Leptospira is a genus of Gram-negative bacteria belonging to the order Spirochaetales and family Leptospiraceae. Pathogenic species are responsible for human and veterinary infection called leptospirosis, which affect mainly tropical and subtropical countries, including Brazil. Knowledge of the pathogenicity mechanisms of leptospires is limited and the vaccine is serovar-dependent. The reverse vaccinology has been applied in the study of outer membrane proteins (OMPs) that have the potential to interact with the host components. In this regard, cell localization assays are important assays for screening of OMPs. LipL31, a protein located in the cytoplasmic membrane of leptospires, may be used as an experimental control in validation of these assays. From the sequenced genome of Leptospira interrogans serovar Copenhageni, the LIC11456 gene was analyzed in silico, and amplified without peptide signal for cloning into the pAE vector using E. coli DH5α. Recombinant LipL31 was expressed in the strain E. coli DE3 Star pLysS with addition of 1 mM IPTG in LB medium and then purified from the soluble fraction by metal affinity chromatography (Ni2 +). The secondary structure of the recombinant LipL31 was analyzed by circular dichroism as a way of evaluating the structural winding, the recombinant presented 70% of its structure composed by α-helix, which is similar to the predicted in silico data. The protein reproduced localization experiments already performed indicating to be internal, and it is suggested that its more stable oligomeric conformation is in the form of dimer.