Expression And Characterization Of Rcombinant Gassericin T Produced By Lactobacillus Gasseri SBT2055

Bacteriocins are antimicrobial proteins or proteinaceous complexes that act mainly against related bacterial species.Bacteriocins from lactic acid bacteria.The bacteriocins from L.acidophilus and L. gasseri, which are commonly found in the human intestine, are expected to have a beneficial effect on human health. A broad-spectral bacteriocin named gassericin T was produced by Lactobacillus gasseri SBT 2055 (from human feces) .We have studied it in prokaryon and eukaryon expression system .To express the bacteriocin named gassericin T with antimicrobial activities in E.coli,DNA fragment encoding the mature peptide of gassericin T were prepared by three rounds of PCR from a total of six 59nt oligoes after modification of the original sequence to the optional codon usage of E. coli and Pichia pastoris,then cloned into pGEX-4T-1. Clones carrying pGEX-4T-1-gat was induced by IPTG. The bacteria were broken down by ultrasonic and the inclusion bodies obtained underwent denaturation , renaturation and purification to obtain GST-gassericinT fusion protein.The antimicrobial activity of the fusion protein was assayed by agar diffusion method and gassericin T demonstrated antimicrobial activities against gram-positive S.aureus,S.typhi,B. subtilis and against gram-negative E. coli,S. viridans,but no effect agaist L. monocytogenes.In order to express gassericin T in its optimium condition, The GAP gene promoter was amplified from P. pastoris GS115 and used to replace the AOX1 promoter(PAOX1) on pPIC9K resulting in plasmid pGAP9K. The gene (called gat gene)was inserted into pGAP9K and transformed into P.pastoris GS115 by electroporation. The high-copy clones were selected by the YPDS plates containing different concentrations of G418. The positive clones, of which the chromosomes were integrated with the gat gene,were identified by PCR. The expression products were tested by Tricine-SDS-PAGE. The results of agar plate cavity diffusion assay and bacteria inhibition analysis indicated that the expression products exhibited the antibacterial activity against S.aureus,Listeria monocytogenes, but no effect agaist E. coli.These data demonstrated that bacteriocins could be expressed in E. coli and Pichia pastoris expression system ,although the result was not exactly the same.The results also indicated a way for its production by bioengineering.