Expression, Purification And Activity Of Tandem Antimicrobial Peptide Bacteriocin LS2 In Chlamydomonas Reinhardtii

Bacteriocin LS2 is one of Bacteriocins isolated from Lactobacillus salivarius with a broad range of inhibitory activity containing a 41 -amino-acid and the molecular mass of the peptide was 4115.1 Da. Bacteriocin LS2 was an extremely pH- and heat-stable peptide with antilisterial activity. Bacteriocin LS2 is a novel member of the class ?d bacteriocins.In our study, three repeats of the Bacteriocin LS2 gene were fused in tandem at first,and inserted into the expression vector pBSK-rbcs2-ble-FMDV2APRO-HA-6xHis-3'UTR.The target gene was transformed into wild-type cc-125 algae using electric conversion method.The Western blotting results confirmed the presence of recombinant antimicrobial peptide Bacteriocin LS2 in four transgenic algal cells which were 2-15 2-16 2-186 2-199 and got that the highest expression of the target protein was 2-186 transgenic algae. And the expression of target protein was stable in 2-186 transgenic algae. The target protein accounted for 0.29% of the Chlamydomonas reinhardtii total soluble proteins.The enrichment of the recombinant antibacterial peptide were achieved by the combination of nickel ions. After vacuum freeze drying, the concentration of the recombinant antimicrobial peptide accounted for 10% of the total soluble protein. The freeze-drying protein was used for the antibacterial activity assays.The tested strains were gram negative bacteria:Salmonella ,Escherichia coli 0157 and gram positive bacteria: Bacillus subtilis 151-1,Listeria monocytogenes. The antimicrobial activity of 3× Bacteriocin LS2 was 117 %,200 %,70 %,123 %.of the positive control (50 ?g/mL gentamicin). The MICs were 90?75?90?75 ?g/mL. The antibacterial peptide 3xBacteriocin LS2 has good thermal stability,acid-base stability and enzyme stability.