Study On Regulation Of PagP Gene Sequence Optimization On The Production Of Recombinant Antimicrobial Peptide

Due to the emergence of antibiotic resistant pathogens around the world,the demand for research anddevelopment of new antimicrobial drugs is increasing day by day.Antibiotic peptide?Antibacteial peptides,AMPs?is a kind of amphiphilic cationic small peptide existing in most organisms which has broad spectrum antibacterial activity and is not easy to produce drug resistance.At present,The genetic engineering techniques are used expressing recombinantly antimicrobial peptides,and it can be carried out in the form of inclusion bodies?Inclusion body,IB?with the fusion label.The protein aggregates formed by over expression of recombinant proteins in Escherichia Coli.This study is based on Metchnikowin?Metch?,Maginin II?Mag II?,Andropin as a target antimicrobial peptide,palmitoylphospholipase?PagP?as fusion label.The experiment makes two aspects of the fusion label transformation.One is to increase the relative content of antimicrobial peptide by shortening the length of fusion label.The second is to mutate proline?Pro?related to the location of fusion tag to leucine?Leu?or isoleucine?Ile?,so as to improve the expression level of recombinant protein and the relative contents of antibacterial peptide.Between tag and antibacterial peptide joining Ni⁺cleavage recognition site,His₆-tag purification label,TEV protease cleavage site.Escherichia Coli expression system was used to structure inclusion body.Using this expression system to express recombinant protein as:P100-NHT-Metch,5-P100-NHT-Met ch,10-P100-NHT-Metch,PagP-NHT-Andropin,5-P100-NHT-Andropin,10-P100-NHT-A-ndropin,127-PagP-NHT-Mag II,135-PagP-NHT-Mag II,121-123-PagP-NHT-Mag II,127-135-PagP-NHT-Mag II,121-121-135-PagP-NHT-Mag II,121-123-121-135-PagP-NH-T-Mag II,10-P100-NHT-Metch?P135L?,5-P100-NHT-Andropin?P135L?.When the inclusion body dissolves,with the Ni⁺cracking fusion protein,the fusion tags PagP and short peptide HT-AMPs separated,fusion tags by corresponding buffer dilution precipitation to remove,short peptide HT-AMPs by Ni-NTA purification,then through TEV enzyme,antibacterial peptides and the fusion tags completely separated,at last we use Carboxymethyl cellulose gel cation exchange column desalination to get pure antibacterial peptides.In this study,the expression conditions of Escherichia coli were 12h and the concentration of inducer IPTG was 0.3 mM,the temperature was 37?.The conditions of Ni⁺cleavage fusion protein were as follows:temperature 60?,target protein concentration was 200?M,the final concentration of Ni⁺was 5¹0 mM,reaction time 24 h,TEV protease enzyme digestion conditions for temperature 25?,the reaction time is 12 h.Through shortening the fusion label of Andropin and Metchnikowin?Metch?,the study finds the protein content increased significantly,and the expression of refactoring carrier pZBR04?pZBR07?pZBR09 content increased by 30.6%?31.4%?39.7%,the expression of refactoring carrier pZBR06?pZBR08?pZBR10 content increased by 49.7%?47.2%?35.3%;After fixed point mutation of Maginin II?Mag II?,the expression of refactoring carrier pZBR17-pZBR22 content increased by 54.4%?60.5%?44.3%?36.7%?17.4%?31.4%.The activity of three kinds of antimicrobial peptides was also tested.Metch could kill Staphylococcus aureus gram positive bacteria;Mag II,Andropin could kill Escherichia coli MG1655 gram negative bacteria and Staphylococcus aureus gram positive bacteria at the same time.This study provides a feasible reference for industrialization of antimicrobial peptides.