Recombinant expression and anti-microbial activity of anti-lipopolysaccharide factor (ALF) from the black tiger shrimp Penaeus monodon.
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, ALFPm3, 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 ALFPm3 which was purified to homogeneity by single chromatography step on expanded-bed Streamline SP6XL. The rALFPm3 was further characterized in terms of N-terminal sequencing and mass spectrometry. Anti-microbial assays demonstrated that rALFPm3 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, rALFPm3 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 ALFPm3 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.
Shrimp Molecular Biology and Genomics Laboratory, Department of Biochemistry, Faculty of Science, Chulalongkorn University, Bangkok 10330, Thailand.
Kunlaya
K
Somboonwiwat
Michael
M
Marcos
Anchalee
A
Tassanakajon
Sirawut
S
Klinbunga
André
A
Aumelas
Bernard
B
Romestand
Yannick
Y
Gueguen
Hélène
H
Boze
Guy
G
Moulin
Evelyne
E
Bachère
D000595:Amino Acid Sequence; D000818:Animals; D000890:Anti-Infective Agents / Q000276:immunology* / Q000494:pharmacology; D001483:Base Sequence; D018076:DNA, Complementary / Q000235:genetics; D005658:Fungi / Q000187:drug effects; D015870:Gene Expression; D006090:Gram-Negative Bacteria / Q000187:drug effects; D006094:Gram-Positive Bacteria / Q000187:drug effects; D006434:Hemocytes / Q000276:immunology; D007447:Invertebrate Hormones / Q000235:genetics* / Q000276:immunology; D008070:Lipopolysaccharides / Q000276:immunology*; D008826:Microbial Sensitivity Tests; D008969:Molecular Sequence Data; D033561:Penaeidae / Q000235:genetics* / Q000276:immunology* / Q000382:microbiology; D010843:Pichia / Q000235:genetics; D011994:Recombinant Proteins / Q000235:genetics / Q000276:immunology / Q000494:pharmacology; D014170:Transformation, Genetic; D014733:Vibrio / Q000187:drug effects / Q000276:immunology / Q000472:pathogenicity
D000890:Anti-Infective Agents; D018076:DNA, Complementary; D007447:Invertebrate Hormones; D008070:Lipopolysaccharides; D011994:Recombinant Proteins