Improving Photocleavage Efficiency of Photocleavable Protein for Antimicrobial Peptide Histatin 1 Expression.

IF 1 4区生物学 Q4 BIOCHEMISTRY & MOLECULAR BIOLOGY

Protein and Peptide Letters Pub Date : 2024-01-01 DOI:10.2174/0109298665276722231212053009

Nana Zhou, Tai An, Yuan Zhang, Guomiao Zhao, Chao Wei, Xuemei Shen, Fan Li, Xiaoyan Wang

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引用次数: 0

Abstract

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.3 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.

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提高光可裂解蛋白的光裂解效率，用于抗菌肽组蛋白 1 的表达。

背景：抗菌肽（AMPs）是抗生素的替代药物，有望克服抗生素耐药性问题。但是，由于抗菌肽对表达宿主的毒性或被宿主体内的肽酶降解，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：为克服对表达宿主的毒性和低表达水平，提供一种新的抗菌肽光可溶性蛋白融合表达方法：方法：通过定向进化和高通量筛选，获得了具有更高光裂解效率的光裂解蛋白突变体 R6-2-6-4。在 R6-2-6-4 基因序列中融合了抗菌肽 Histatin 1 的 DNA 编码序列。融合基因在大肠杆菌表达系统中成功表达：结果：在 PhoCl 突变体 R6-2-6-4 中融合抗菌肽 Histatin 1 可成功表达和纯化。在5 L发酵罐中扩增后，PhoCl突变体（R6-2-6-4）-Histatin1在发酵罐中的表达量提高到87.6 mg/L，光裂解得到的Histatin1也可达到11 mg/L。制备的 Histatin1 粉末在 4oC 温度下保存 4 个月仍保持稳定，未发生任何降解。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具有一定的通用性：结论：抗菌肽Histatin 1、β -Defensin105和Lysostaphin通过光裂解蛋白突变体成功表达和纯化。这为在大肠杆菌表达系统中表达和纯化抗菌肽提供了一种新策略。

本文章由计算机程序翻译，如有差异，请以英文原文为准。

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来源期刊

Protein and Peptide Letters 生物-生化与分子生物学

CiteScore

2.90

自引率

0.00%

发文量

审稿时长

2 months

期刊介绍： Protein & Peptide Letters publishes letters, original research papers, mini-reviews and guest edited issues in all important aspects of protein and peptide research, including structural studies, advances in recombinant expression, function, synthesis, enzymology, immunology, molecular modeling, and drug design. Manuscripts must have a significant element of novelty, timeliness and urgency that merit rapid publication. Reports of crystallization and preliminary structure determination of biologically important proteins are considered only if they include significant new approaches or deal with proteins of immediate importance, and preliminary structure determinations of biologically important proteins. Purely theoretical/review papers should provide new insight into the principles of protein/peptide structure and function. Manuscripts describing computational work should include some experimental data to provide confirmation of the results of calculations. Protein & Peptide Letters focuses on: Structure Studies Advances in Recombinant Expression Drug Design Chemical Synthesis Function Pharmacology Enzymology Conformational Analysis Immunology Biotechnology Protein Engineering Protein Folding Sequencing Molecular Recognition Purification and Analysis