Synthsis, purification and characterization of peptides derived from E.coli alpha hemolysin

F. GUZMÁN; S.MATÉ; L. BAKÁS; V. HERLAX

Congreso; XLII Reunión Anual de SAB; 2014

Escherichia coli alpha hemolysin (HlyA) is a pore-forming protein which belongs to the family of 'Repeat in toxins'(RTX). On the basis of experimental data and structural predictions, four peptides derived from HlyA were synthesized by the solid phase peptide synthesis method, using the Fmoc strategy. The four peptides were design as follows: PEP 1: correspond to transmembrane domain that it was described as hemolytically active; PEP 2: also a transmembrane domain which sequence correspond to a cholesterol recognition/interaction amino acid consensus domain (CARC); PEP3: similar to PEP2 but 6 aminoacids were added in the amino extreme and PEP4 correspond to a CARC sequence located near the acylation sites. The aims of this work were to look for a peptide which present hemolytic activity, and study the participation of CRAC and CARC in the stabilization of HlyA monomers in membranes by their interaction with cholesterol. After peptide synthesis, they were purified by Reverse-phase high performance liquid chromatography (HPLC), using C-18 column. The molecular mass of the peptides were determined by mass spectrometry (MS). Finally, peptide structure was determined by circular dichroism (CD). It is important to mention that the addition of 6 aminoacids to PEP 2 (PEP3) gives the peptide a more organized structure. The hemolytic activity of peptides was measured using human erythrocytes and inhibition of hemolytic activity assays were performed pre-incubating erythrocytes with peptides and then adding them to wild type toxin. Results obtained for PEP 2 are promising and encourage us to use it in the design of immunotoxins.