Expression, Purification And Characterization In Vitro Of Antibacterial Peptide Cecropin D

Antibacterial peptides are a kinds of small molecule peptides.They are produced by specific gene encoding,when organisms are invaded by pathogen outside.Compared to traditional antibiotics,antibacterial peptides have many characteristics including low molecular weight,soluble in water,thermal stability,broad-spectrum antibiotic,difficult to develop drug resistance,and so on.They show a good clinical application perspective.The naturally occurring antibacterial peptides content in vivo is very low and difficult to isolate.And the cost of chemical synthesis is very high.It is difficult to satisfy demands of basic research and clinical therapy.In view of these problems,it is quite important for producing antibacterial peptides by the methods of gene engineering.Because antibacterial peptides can kill bacterial and they are unsuitable to express directly in prokaryote cell system,So we can only use fusion expression in prokaryote or secreting expression in yeast.In the present study,we cloned,expressed,purified and characterized of antibacterial peptide Cecropin D in vitro.Firstly,according to the prefferred codons of E.coli,6 oligonucleotides were chemically synthesized,phosphorylated,annealed,linked and cloned into pThioHisA vector.After optimizing the terms of expression,the fusion protein pThioHisA-eCD was highly expressed in E.coli BL21 and it accounted for 15%of total protein.The fusion protein was purified by CM-sepharose Fast Flow and its purity was over 90%. And it was inoculated into guinea pigs to produce antiserum.Secondly,a gene for yCD using prefferred codons by the yeast Pichia pastoris was synthesized.This synthesized gene was fused withαfactor signal peptide gene of Saccharomyces cerevisiae and the fused geneα-yCD was cloned into yeast expression vector pPIC9K.The recombinant plasmid pPIC9K-α-yCD was linearized by Sal I,transformed into Pichia pastoris GS115 by electroporation,and stable multicopy integrants were screened in medium containing different concentrations of G418.We have acquired four multicopy integrants from two hundred.The positive clones,of which the chromosomes were integrated withα-yCD gene,were identified by PCR and induced by 0.5%methanol.The expression product was purified by CM-sepharose exchange chromatography.Tricine-SDS-PAGE demonstrated specific protein band with a relative molecular weight of 3.gKD and the purity of Cecropin D was over 90% after purification.It was showed by Western blot that the target protein Cecropin D had remarkable immunoreactivity with antiserum produced by fusion protein pThioHisA-eCD.Agarose diffusion assay and liquid phase analysis indicated that antibacterial peptide Cecropin D has the antibacterial activity on some Gram-positive bacterium and Gram-negative bacterium.In addition,antibacterial peptide Cecropin D showed an extreme thermal stability,when it was boided 30min.In a word,We obtained fusion protein pThioHisA-eCD and secreting protein Cecropin D with high expression and puritification.We had preliminary research on antibacterial activity in vitro of Cecropin D which was expressed by Pichia pasroris.These results were helpful for futher research on express level,antibacterial efficiency,meachanism of antibacterial peptides.It also established foundation for mass production and development of new antibiotics.