Recombinant Expression, Purification And Activity Determination Of Antimicrobial Peptide Hydramacin-1 In Pichia Pastoris

Hydramacin-1, an antibacterial peptide, was isolated and identified in the study of the epithelium defense process of Hydraacin-1 by Jung S,with a size of about 7 KDa and 60 amino acid residues. In conclusion, Hydramacin-1 has the advantages of strong cationicity,stable structure, broad antimicrobial spectrum and strong Bacteriostatic ability. It has great application value.In this study,the DNA sequence of Hydramacin-1 was optimized according to the codon preference of Pichia pastoris. It was ligated to the Pichia pastoris expression vector pPICZaA and transformed into Pichia pastoris GS115. The expression of the positive transformants was optimized, and the optimal conditions which the total protein expression reached 160?g / mL were as follows: The final concentration of methanol was 1.5% and the induction time was 120 h. The supernatant of the fermentation broth was purified by cation exchange column, and the target protein was successfully obtained. The MIC of Hydramacin-1 against E. coli 0157 (ATCC 35150) was 80?g/mL. The MIC value of S.aureus (ATCC 25923) was greater than 100 ?g/mL,It is less sensitive to Gram-positive bacteria.We further investigate whether the changes of positive charge, hydrophobicity and other factors can regulate its activity by amino acid replacement. We designed six mutants,namely Q17P, N28H, D29S, E33R, E42V and E43VE43V. The mutant plasmids were obtained by point mutation PCR, then transformed into Pichia pastoris GS115 for expression induction.The expression was induced in the yeast strain GS115. Finally, the physicochemical parameters and antimicrobial activities of different mutants and wild type were compared. The results showed that The results showed that the antibacterial activity of Q17P on S. aureus and The antibacterial activity of N28H and D29S on E. coli 0157 was improved. This is because the hydrophobicity of Q17P increased, N28H replaced with homologous amino acids, D29S increased by 1 positive charge. E42V has high hydrophobicity and low antibacterial activity, E33R increased two positive charges, and the antibacterial activity was also significantly reduced. E42VE43V has the highest positive charge and hydrophobicity, and its antimicrobial activity is the worst among six mutants. It can be seen that increasing the positive charge and hydrophobicity in a certain range can increase the antibacterial activity of the antibacterial peptide Hydramacin-1, but beyond a certain threshold, it will result in the decrease of antibacterial activity.