Prokaryotic Expression, Purification Of Human Beta Defensin 4 In E.Coli And Initial Study On Pseudomonas Aeruginosa

Pseudomonas aeruginosa (P. aeruginosa, PA) distributed widely in nature, and was of multi-drug resistance ability and high adaptive to environment. It was an opportunistic pathogen to human being, and frequently isolated from patients suffering from bacteremia in trauma, burn and immunity-compromised hosts, so it was seriously threaten to human health. It was one of the most important pathogens in the hospital infection. In particular, P. aeruginosa was a difficult pathogen to eradicate because of its formation of biofilm and resistance to a wide range of antibiotics. The emergence of antibiotic resistant organisms had been attributed to over use of broad spectrum antibiotics in humans. The antibiotic resistant P. aeruginosa strains are not susceptible to conventional antibiotics. In view of the urgent present situation, how to find a more effective medicine to control the infection, simultaneously reducing the medicine toxicity and preventing the drug resistance has become the most difficult problem in the world.Human defensins are the first-line effector moleculars of innate immunity. They are cationic peptides containing 40-50 amino acid residues. The peptides contain 6 conserved cysteines linked in disulfide bonds that stabilize the molecules as triple-stranded amphiphilic -sheet structures. In vitro, human defensins exhibit antimicrobial activity against some bacteria, fungi, enveloped viruses, and parasites. A few of these peptide antibiotics have entered clinincal trials with success to date. Human Beta defensin 4 was founded by Wolf-Georg Forssmann in 2001, containing 50 amino acids with broad antibacterial, especially to P. aeruginosa. Yanagi research showed that all patients with infection caused by mucoid P. aeruginosa had detectable hBD2 and hBD4 levels. In-vitro colony count assays showed a potential synergism between hBD2 and hBD4 in inhibiting bacterial proliferation, and hBD4 showed higher activity. The findings indicate that hBD, especially hBD2 and hBD4, are pathophysiologically important in infections caused by mucoid strains of P. aeruginosa. The hBD4-engineered cells were infected with Pseudomonas aeruginosa, a type of bacteria commonly found in hospitals, and allowed to incubate. Analysis revealed that the genetically altered cells containing hBD4 were more resistant to microbial infections than the unaltered cells. The natural production of human defesin 4 is low, and the cost of chemical synthesis is expensive. The use of recombinant expression methods might be introduced to obtain hBD4 in a quantity sufficient to meet the demand for it in the research on mechanism and the development of new drugs.Due to their natural destructive behavior toward microorganisms, fusion strategy has been utilized for the expression of small antimicrobial cationic peptides in E. coli to mask the toxicity of the product to the host cell and to protect the small peptides from proteolytic degradation. Escherichia coli expression system was one of the most successful systems for protein expression; many cationic peptides have been successfully expressed in this system. pET system was choosed for the human defensin 4 expression. The GST fusion tag was added to N terminal of hBD4 for soluble expression. The enterokinase cleavage site was added between the fusion protein and hBD4 to obtain the native sequence of hBD4 by enterokinase cleavage and Ni-NTA purification. The mostly contents and results are as follows:Firstly, reconstructed the prokaryotic expression vector and optimizing expression target protein: The target gene was selective amplified by PCR using pET42 recombinant plasmid as source of thehBD4 gene, doubled digested with restriction endonucleases and cloned into correspondingly digested expression vector pET-42a(+) to generate the recom binant plasmid pET42-hBD4, The correct construct was confirmed by DNA sequencing and named pET42-hBD4. The pET system is the most powerful system yet developed for the cloning and expression of recombinant proteins in E. coli. Target protein expression is induced by providing a source of T7 RNA polymerase in the host cell. Another important benefit of this system is its ability to maintain target genes transcriptionally silent in the uninduced state. Target genes are initially cloned using hosts that do not contain the T7 RNA polymerase gene, thus eliminating plasmid instability due to the production of proteins potentially toxic to the host cell. Then the plasmid was transferred into an expression host (E.coli BL21 (DE3)) containing a chromosomal copy of the T7 RNA polymerase gene under lacUV5 control, the target protein expression is induced by the addition of IPTG to the bacterial culture. Optimization of the expression conditions revealed that 4h induction and growth at 37℃and the IPTG concentration is 0.5 mmol/L.Secondly, purification of recombinant GST-hBD4 fusion protein. The protein was purified by immobilized metal ion affinity chromatography (IMAC). The advantage is that the mental ion ligand has a good stability and a big adsorption quantity, low cost and short purification time. The purity of recombinant fusion protein was about 90% after Ni-NTA purification. The hBD4 peptide was separated from the GST tag by Ni-NTA purification after Enterokinase digestion. The purity is about 95%, satisfied with the requirement for next experiments.Thirdly, the evaluation of the bactericidal activity of recombinant hBD4 in vitro. The purified protein had antibacterial activities to P. aeruginosa(ATCC27853), Staphyloccocus aureus(ATCC25923) Escherichia coli(ATCC35218) and Enterococcus faecalis(ATCC29212) determined by inhibition zone assay. The minimal inhibitory concentrations (MIC) and the minimal bactericidal concentrations (MBC) of the protein were assayed. The results showed that the antibacterial activities of recombinant hBD4 is inferior to piperacillin and ciprofloxacin, but superior to imipenem, which indicated that hBD4 are more efficient than the two traditional antibiotics on P. aeruginosa in vitro.In conclusion, we have constructed the prokaryotic expression vector pET42-hBD4 successfully. The recombinant plasmid was transferred into an expression host and obtained good yields of recombinant protein. The recombinant hBD4 protein exhibits its antibacterial activities to P. aeruginosa. Our findings will bring our research work to be promising.