Influence Of Disulfide Bonds On Antimicrobial Activity And Tandem Multimers Expression In Pichia Pastoris Of Plectasin

Plectasin is a 40-amino acid residue fungal defensin. It has strong antimicrobial activity against gram-positive bacteria, especially against Streptococcus pneumoniae, including strains resistant to conventional antibiotics. In vitro, S. pneumoniae was killed rapidly by plectasin at rates comparable to both vancomycin and penicillin. In addition, plectasin did not exhibit hemolytic activity toward human erythrocytes, which possess of the potential as beneficial therapeutics in overcoming infectious diseases.The method that aims to explore the influence of disulfide bonds to the antimicrobial activity of plectasin. The recombinant vectors with plectasin derivatives that have already successfully constructed before were expressed in P. pastoris X-33. Tricine SDA-PAGE analysis proved that all of the plectasin derivatives were successfully expressed. The plectasin derivatives were purified by Sephadex G-25 column. The antimicrobial activity of the peptides was identified. The result showed that different disulfide bonds have different effects on antibacterial activity of plectasin. The disulfide bond C15-C37 has the largest effect on plectasin than the other two disulfide bonds, for the mutants (C15/37A)Plectasin and (C4/15/30/37A)Plectasin almost has no antimicrobial activity. The disulfide bond C19-C39 has smaller effect on plectasin, since all C19-C39 disulfide mutunts has antimicrobial activity, but a bit lower than plectasin.The tandem multimeric expression of various defensin could increase the expression level, and the polypeptide could be steadier as the form of multimers. In this study, the tandem multimers of plectaisn were constructed and expressed in Pichia pastoris.First, the monomer plectasin was successfully expressed in P. pastoris X-33. The cleavage sites of the isocaudamer XbaⅠ/NheⅠcoding sequence were added to the terminals of the optimized plectasin for the construction of the multimers. In addition, the formic acid and hydroxylamine cleavage sites were also added between plectasin and the vector protein respectively. The genes were synthesized and inserted into the vector pPICZαA, and then transformed and induced by methanol. Following 120 h induction in P. pastoris, the amount of total secretion protein of recombination strains X-33(pPICZαA/PPD) and X-33(pPICZαA/PN) reached 339μg/mL and 307μg/mL, respectively. The monomer PPD and PN were active against Staphylococcus aureus after purified and then cleaved by formic acid or hydroxylamine, which layed a foundation of the construction of plectasin multimers.Base on the analysis of the moner expression experiment, plectasin multimers were constructed using the isocaudamer XbaⅠ/NheⅠ. Digested the recombinant vectors by XbaⅠ/NheⅠand XbaⅠrespectively. Linear vectors and the insert fragments with same cohesive ends were connected by T4 DNA ligase. Plectasin dimer and tetramer were constructed using the same method. The multimers were induced and expressed in Pichia pastoris X-33. Tricine SDA-PAGE analysis revealed that the dimer was successfully expressed, and the tetramer was not detected. So the expression parameters need to be studied.In conclusion, the comparison of the antibacterial activities of the plectasin disulfide bonds derivatives revealed that disulfide bonds played an important role in maintaining the maximal activity. And then we successfully constructed the monomer and the dimer recombinant vectors and expressed in P. pastoris X-33, which lay a foundation for its production and application .