Expression and partial purification of anti-lipopolysaccharide factor of black tiger shrimp Penaeus monodon

Abstract

Anti-lipopolysaccharide factor (Anti-LPS factor) is one of the major protein in the crustacean immune system. In horseshoecrab, this protein has an antibacterial effect on the growth of some Gram-negative bacteria. It can bind and neutralize bacterial endotoxin, lipopolysaccharide (LPS). In the black tiger shrimp (Penaeus monodon), a cDNA encoding anti-LPS factor has been isolated from the hemocytes cDNA library. It contains an open reading frame of 372 bp coding for 123 aa residues with a predicted molecular weight of 13.7 kDa. In this study, NH₂₋ terminal truncated derivative of anti-LPS factor, were cloned and expressed in baculovirus and yeast Pichia pastoris expression system. The bacovirus expression system did not yield any recombinant protein however, in Pichia pastoris expression system, expression of recombinant protein was observed. Four clones were found to have high expression of recombinant protein, which have inhibitory effect against Escherichia coli over 30%. The highest expression level was found in clone number 12, with 52.7% inhibition from crude culture medium. Clone number 12 was used for production of the recombinant protein in large quantity for further purification. The purification was performed by affinity column chromatography, which purified the protein by 65.45 folds, with 40.9% recovery, 99.4% of others protein was removed in this step. Further purification was perform by gel filtration column chromatography which purified the protein further 12.5 folds, with a 2.0% recovery. The partial purified recombinant protein was tested for antibacterial activity against 4 strains of bacteria. The addition of 10 µg of partially purified anti-LPS protein can inhibit Vibrio harveyi, Escherichia coli, Staphylococcus aureus and Micrococcus luteus at 87.7, 81.7, 14.3 and 39.8% respectively.