Purification Process Andactivity Analysis Of Recombinant Protamine

BackgroundAntimicrobial peptides(AMPs)are a class of short peptides produced by a variety of organisms through the immune system which are of low molecular,cationic protein,linear or spiral hydrophilic sequence.Antimicrobial peptides with wide antimicrobial spectrum can destroy the structure of cell membrane to achieve antimicrobial effect.Protamine protein(PP)is one of the hotpots of antimicrobial peptides.It is rich in Arginine small molecule nucleic acid protein and can replace Histidine and combined with DNA.The protamine is the only registered antidote of heparins in cardiac operation.Cytoglobin is a new leading member of the globin family.It was discovered in 2001 and named cytoglobin in 2002.As the Cytoglobin has a high expression in the E.coli system,the project hope to realize the efficient production of target protein with the fusion Cytoglobin.Objective(1)This project aimed to study the production of protamine by genetic engineering methods.(2)In order to find the optimal expression system of protamine,this project use two different expression systems.(3)This project aimed to optimize the purification process and realized the industrial production of protamine.Method(1)The protamine was produced by genetic engineering methods in the E.coli Expression Systems and Baculovirus-Insect Expression System.(2)The project uses Cytoglobin-Protamine fusion protein to produce the protamine in the E.coli Expression Systems.(3)Using CNBr to cut the cleavage sites of methionine between Cytoglobin and Protamineinthesubsequentpurificationprocess.Bysite-directed mutation,the gene of Cytoglobin is not included the site of Met(except initiation codon).Conclusion(1)Site-directed mutation and expression of CYGBm has completed successfully.(2)Construction of recombinant plasmid pET22b-CYGBm-PP,expression of CYGBm-PP and activity analysis ofrecombinant protamine.(3)The results of antibacterial experiments show that the recombinant protamine has antibacterial activity.(4)The yield of recombinant protamine is 17.9%.(5)Construction of recombinant plasmid of pFastBacT1-PP-His-LIR-DsRed2 and pFastBacT1-His-PP-LIR-DsRed2 have completed successfully.