Expression, purification and functional study of mycobacteriophage D29 histidine-asparagine-histidine endonuclease

IF 1.4 4区生物学 Q4 BIOCHEMICAL RESEARCH METHODS

Protein expression and purification Pub Date : 2025-06-02 DOI:10.1016/j.pep.2025.106753

Bo Fu, Lvming Wu, Xin Wang, Hongbin Sun

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引用次数: 0

Abstract

Mycobacteriophage histidine-asparagine-histidine endonuclease (mpHNHE) is a protein encoded by mycobacteriophage D29, featuring a conserved HNH motif and belonging to the HNH nuclease superfamily. To explore its physiological functions, the recombinant plasmid pET-28a (+)-mpHNHE was constructed and expressed in E. coli BL21 (DE3). The inclusion body form of the expression product was purified using urea denaturation combined with nickel affinity chromatography and gel filtration chromatography. Structural characterization revealed that mpHNHE exists as a monomer in solution, predominantly composed of β-sheets, and exhibits good structural stability. Enzymatic property studies indicated that mpHNHE has high nuclease activity, significant substrate size selectivity, and metal ion dependence. These findings not only provide new insights into the structure-function relationship of HNH-type nucleases but also provide a molecular basis for the development of new nuclease tools and lay the foundation for understanding the mechanism of mpHNHE in D29.

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分支噬菌体D29组氨酸-天冬酰胺-组氨酸内切酶的表达、纯化及功能研究。

分枝噬菌体组氨酸-天冬酰胺-组氨酸内切酶（mpHNHE）是一种由分枝噬菌体D29编码的蛋白，具有保守的HNH基序，属于HNH核酸酶超家族。为探究其生理功能，构建了重组质粒pET-28a(+)-mpHNHE，并在大肠杆菌BL21（DE3）中表达。采用尿素变性结合镍亲和层析和凝胶过滤层析对表达产物的包涵体形态进行了纯化。结构表征表明，mpHNHE在溶液中以单体形式存在，主要由β-片组成，具有良好的结构稳定性。酶学性质研究表明，mpHNHE具有高核酸酶活性，显著的底物大小选择性和金属离子依赖性。这些发现不仅为hnh型核酸酶的结构-功能关系提供了新的见解，而且为开发新的核酸酶工具提供了分子基础，并为理解mpHNHE在D29中的作用机制奠定了基础。

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 expression and purification 生物-生化研究方法

CiteScore

3.70

自引率

6.20%

发文量

120

审稿时长

32 days

期刊介绍： Protein Expression and Purification is an international journal providing a forum for the dissemination of new information on protein expression, extraction, purification, characterization, and/or applications using conventional biochemical and/or modern molecular biological approaches and methods, which are of broad interest to the field. The journal does not typically publish repetitive examples of protein expression and purification involving standard, well-established, methods. However, exceptions might include studies on important and/or difficult to express and/or purify proteins and/or studies that include extensive protein characterization, which provide new, previously unpublished information.