Résumé
Anti-lipopolysaccharide factors (ALFs), originally characterized from horseshoe crabs, have been recently identified from hemocytes of the black tiger shrimp,
Penaeus monodon, by a genomic approach. In order to characterize the properties and biological activities of this immune effector in shrimp, ALF
Pm3, the most abundant isoform found in
P. monodon, was expressed in the yeast
Pichia pastoris. Large-scale production in fermentor provided 262
mg/l of recombinant ALF
Pm3 which was purified to homogeneity by single chromatography step on expanded-bed Streamline SP6XL. The rALF
Pm3 was further characterized in terms of N-terminal sequencing and mass spectrometry. Anti-microbial assays demonstrated that rALF
Pm3 has a broad spectrum of anti-fungal properties against filamentous fungi, and anti-bacterial activities against both Gram-positive and Gram-negative bacteria, associated with a bactericidal effect. Interestingly, rALF
Pm3 is highly efficient against various
Vibrio species including strains pathogenic for shrimp. Finally, a synthetic peptide corresponding to a part of the putative LPS-binding site of ALF
Pm3 was shown to display activities mainly directed against Gram-positive bacteria indicating the involvement of the full molecule to the anti-microbial activity for Gram-negative bacteria.