Background:
Antimicrobial peptides (AMPs) are promising alternative agents for
antibiotics to overcome antibiotic resistance problems. But, it is difficult to produce large-scale antimicrobial research due to the toxicity towards expression hosts or degradation by peptidases in
the host. Therefore, heterologous recombinant expression of antimicrobial peptides has always
been a challenging issue.
Objective:
To overcome toxicity to the expression host and low expression level, a new photocleavable protein fusion expression method for antimicrobial peptides is provided.
Methods:
Through directed evolution and high throughput screening, a photocleavable protein mutant R6-2-6-4 with a higher photocleavage efficiency was obtained. The DNA coding sequence of
antimicrobial peptide Histatin 1 was fused within the sequence of R6-2-6-4 gene. The fusion gene
was successfully expressed in Escherichia coli expression system.
Results:
Antimicrobial peptide Histatin 1 could be successfully expressed and purified by fusing
within PhoCl mutant R6-2-6-4. The antimicrobial activity was rarely affected, and the MIC value
was 33 ug/mL, which was basically equivalent to 32 ug/mL of the chemically synthesized Histatin
1. After amplification in a 5 L fermenter, the expression of PhoCl mutant (R6-2-6-4)-Histatin1 improved up to 87.6 mg/L in fermenter, and Histatin1 obtained by photocleavage also could up to 11
mg/L. The prepared Histatin1 powder remained stable when stored at 4oC for up to 4 months without any degradation. In addition, the expression and photocleavage of β -Defensin105 and Lysostaphin verified the certain universality of the PhoCl mutant fusion expression system.
Conclusion:
Antimicrobial peptides Histatin 1, β -Defensin 105 and Lysostaphin were successfully expressed and purified by photocleavable protein mutant. This may provide a novel strategy to
express and purify antimicrobial peptides in the Escherichia coli expression system.