Screening,Expression,Purification And Characterization Of Antimicrobial Peptides

With the discovery and application of penicillin in 1929,more and more antibiotics were discovered or developed and benefited modern medicine.However,with the overuse of antibiotics,not only are various infections cured,but there is also an increasing number of multidrug resistant(MDR).Shortly after the use of penicillin in clinical medicine,the abuse of antibiotics gradually appeared;the resistance of many pathogens increased,and many super bacteria were continuously isolated and discovered,such as methicillin-resistant Staphylococcus aureus(MRSA)which can withstand methicillin.MRSA exhibits resistance to all β-lactams and other antibiotics,which makes clinically limited treatment options for the infections it causes.Clinically,the use of antibiotics or a combination of multiple antibiotics for the treatment of MRSA infection has not only achieved little effect,but also has increased the resistance of MRSA.On the one hand,there is a growing lack of methods for treating MRSA infections.On the other hand,the resistance of MRSA is increasing,and this super bacteria continues to pose a threat to human health.Studies have shown that the threat posed by super bacteria can be alleviated or even cured by small antimicrobial peptides.Recently,the trend of use of small peptides as drug candidates has proliferated because they offer significant advantages over small molecule drugs.Antimicribial peptides have the advantages of small relative molecular weight,good heat resistance,broad-spectrum antimicrobial properties,and no drug resistance.They are promising and clinically valuable in the face of the threat of antibiotic resistance.This experiment describes a method for rapid screening,expression and purification of antimicrobial peptides.The antimicrobial peptide gene was fused to a heat-resistant CL7 tag by SLOPE method and cloned into E.coli expression vector pET23a and Pichia pastoris expression vector pHBM905BDM and pPICZα.In this study,20 E.coli expression vectors and 10 Pichia pastoris expression vectors were constructed.The CL7 tag can also provide heat resistance to the fusion protein,and the impurities in the supernatant can be initially removed after high temperature treatment.After Ni-NTA purification,the fusion protein was digested with high-efficiency and low-cost HRV 3C protease on a nickel column,and the antimicrobial peptide was released and further purified to obtain the desired antimicrobial peptide.Among them,5 antimicrobial peptide were screened and purified from the E.coli expression system,and 1 antimicrobial peptide from the Pichia pastoris expression system.After the inhibition zone test and MIC detection,two kinds of antimicrobial peptides with antimicrobial activity were successfully screened and purified.The expression vector was rapidly constructed by synthesizing the target protein in combination with the SLOPE method during the construction of the expression vector.The purification process simplifies the purification process and reduces the purification cost by utilizing the heat resistance of the antimicrobial peptide and the fusion protein parter CL7 tag.The use of E.coli expression system in combination with Pichia pastoris expression system also increases the universality of this method.