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. Objectives: 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.

中文翻译：

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提高光裂解蛋白对抗菌肽 Histatin 1 表达的光裂解效率

背景：抗菌肽（AMP）是有前途的抗生素替代药物，可以克服抗生素耐药性问题。但由于对表达宿主的毒性或宿主体内肽酶的降解，很难进行大规模的抗菌研究。因此，抗菌肽的异源重组表达一直是一个具有挑战性的问题。目的：克服对表达宿主的毒性和表达水平低的问题，提供一种新的抗菌肽光裂解蛋白融合表达方法。方法：通过定向进化和高通量筛选，获得具有更高光裂解效率的光裂解蛋白突变体R6-2-6-4。抗菌肽Histatin 1的DNA编码序列融合在R6-2-6-4基因的序列内。该融合基因在大肠杆菌表达系统中成功表达。结果：抗菌肽Histatin 1可以通过与PhoCl突变体R6-2-6-4融合来成功表达和纯化。抗菌活性很少受到影响，MIC值为33 ug/mL，基本相当于化学合成的Histatin 1的32 ug/mL。在5 L发酵罐中扩增后，PhoCl突变体（R6-2）的表达-6-4)-Histatin1在发酵罐中提高至87.6 mg/L，通过光裂解获得的Histatin1也可提高至11 mg/L。制备的 Histatin1 粉末在 4℃ 下保存长达 4 个月保持稳定，没有任何降解。此外，β-Defensin105和Lysostaphin的表达和光裂解验证了PhoCl突变体融合表达系统的一定普适性。结论：光裂解蛋白突变体成功表达并纯化了抗菌肽Histatin 1、β-Defensin 105和Lysostaphin。这可能为在大肠杆菌表达系统中表达和纯化抗菌肽提供新的策略。