Expressão do peptídeo recombinante Defb-Lm02, uma β-defensina da serpente Lachesis muta

Abstract

Antimicrobial peptides (AMPs) are components of innate immune system, are present in all living organisms, acting quickly and in unspecific way as a chemical barrier and they have antimicrobial activity. β-defensins is a major family of AMPs with characteristic β- sheet-rich fold, six conserved Cys with particular spacing and intramolecular bonds. They are extensively studied in mammals but scarcely in snakes. The β-defensin Defb-Lm02, of Lachesis muta snake, is codified by a gene structured in three exons and has 38 amino acid residues, molecular weight of 4.6 kDa, net charge of +8 at pH 7 and isoelectric point (pI) of 10.35. The synthetic linear peptide of Defb-Lm02 was tested to antimicrobial activity: it presented the minimum inhibitory concentration against Gram negative bacteria: Escherichia coli 16 μg/mL and Citrobacter freundii 16 μg/mL; and Gram positive: Micrococcus luteus 4 μg/mL and Staphylococcus aureus 32 μg/mL. It did not inhibit the growth of Gram-negative Klebsiella pneumonia, Serratia marcescens, Providencia rettgeri and Morganella morganii until the concentration of 64 μg/mL. These results may be due to the linearity of synthetic peptide. As the defense tasks of β-defensin could be dependent on their 3D structure, the production of recombinant β-defensin may potentiate its antimicrobial activity or suggest a new function. The synthetic gene, maltose binding protein (MBP) – TEV protease recognition site – DefbLm02, was cloned into the pET28a expression vector. The protein was expressed in E. coli BL21 Star and C43, at two temperatures 30 ° C and 37 ° C. After 4 hours of induction with IPTG 1 mM, which showed the best condition of expression was BL21 Star at 30 °C. The major part of fusion protein was soluble after purification by affinity chromatography and after proteolysis. The protein fusion was checked by Western blot using an antibody anti-MBP.