| Infection is a common complication of massive burns and the sepsis arises from these infection is one of the major reasons for multi-organ failure or even death. In recent years, treatment of infections with traditional antibiotics has been challenged by the increasingly severe problem of antibiotic-resistant bacterial strains, which has, at least partly, resulted in a constantly high mortality in burned patients. In fact, the drug-resistant strains have entangled not only burns surgeons but literally the whole medical science. Thus, it's of great importance to find new type antibiotics that illicit no drug-resistance. Previous studies have demonstrated that antimicrobial peptides, as part of the host defense system, display extensive microorganism killing activities. Natural proteins and eliciting no drug-resistance, these peptides are potential candidate to be developed into new type antibiotics.Defensins are a cluster of antimicrobial peptides extensively present in our body with a broad spectrum of antimicrobial activities but will not cause drug-resistance. Amongst is human beta-defensin 3 (hBD3), which demonstrates antimicrobial activity against a vast variety of microorganisms like both Gram-positive and Gram-negative bacteria and even funges. It's especially potent to Gram-positive strains including Staphylococcus aureus. In addition to the well-recognized antimicrobial properties, recent reports picture this protein as potent immunomodulators, virus and tumor cell killer. In addition to high bioactivity stability like other β-defensins in high temperature and extreme pH environments, hBD3 is salt-insensitive. These favorable physical and chemical properties, together with its attractive biological activities, make hBD3 promising target for drug development. Finally, it's worth mention that gene-engineered hBD3 in E.coli shares the same biological activities with its natural or syntheticcounterparts. This further indicates that hBD3 may carry great hope for development of a new type of antibiotic--peptide antibiotic, and provide a powerful solution for current bacterial antibiotic-resistance problem.Since hBD3 is positively charged, protein fusion strategy is very popular for engineering of hBD3 in previous reports. In this way scholars aim to to neutralize the positive charges and thus attenuate its host toxicity. However, in these studies, the expression systems are unexceptionally prokaryotic, and the fusion protein is produced in the form of inclusion bodies, which makes the subsequent processes more complicated. As a result, this will come to a low productivity, unable to meet to the cost-product balance for drug development.Based on our experience in hBD3 gene engineering in E.coli and other groups' experience in gene engineering of other defensins in yeast expression system, the present study adapts a new strategy for hBD3 gene engineering by fusing the potent Gram-positive bacteria killing peptide hBD3 with the bioactive 35-211 aa of a potent Gram-negative protein killing peptide, bactericidal/permeability increasing protein (BPI) , and express the fusion protein in pastoris picha yeast expression system. This system has a larger genome capacity and generally gives higher productivity. The foreign protein is more likely to be expressed in soluble form and secreted into the supernatants and thus facilitates the subsequent purification procedures.We successfully constructed the yeast expression vector pICZαB-hBD3-BPI, and the recombinant X-33/pICZαB-hBD3 -BPI clones demonstrated target fusion protein expression in supernatants after methanol induction for 96 hours. Western blot analysis confirmed that the recombinant hBD3-BPI (rhBD3-BPI) protein product was positive for both hBD3 and BPI. rhBD3-BPI accounts for 15.6% of the whole protein in supernatant. After purification by hydrophobic chromatography and cation exchange chromatography, the final productivity was 5.48mg/L, with a recollection rate of 68.5% and a purity of 89%.Bioactivity analysis indicates that rhBD3-BPI displayed potent antimicrobial activity against a wide spectrum of bacteria, including both Gram-positive andGram-negative strains, despite of standard or clinically isolated wild-type, antibiotic-resistant or not. Its minimal inhibitory concentration(MIC )is l-8μg/ml, minimal bactericidal concentration (MBC ) is 4-16μg/ml. In the meanwhile, like BPI, the rhBD3-BPI protein can also neutralize LPS activity. Additionally, like hBD3, rhBD3-BPI is also salt-insensitive. No significant discont of antimicrobial activity was observed at 150mmol/L-200mmol/L NaCl concentrations.The present study for the first time expressed hBD3 in pastoris picha yeast expression system. And also for the first time, hBD3 was fused with BPI for fusion expression. The fusion protein was secreted into the supernatant after methanol induction culture and the purified rhBD3-BPI demonstrated potent antimicrobial activity against both Gram-positive and Gram-negative strains. It's also salt-insensitive and can neutralize LPS activity. So, in summary, the fusion expression of hBD3-BPI in pastoris picha yeast expression system is feasible and the fusion strategy has extended the biological activities in addition to antimicrobial spectrum, thus potentially increased the potential clinical applications and provided useful clues for gene engineering and drug development of hBD3.
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