Cloning and characterization of a novel nitric oxide synthase gene from Exiguobacterium profundum and its expression in Escherichia coli

IF 1.4 4区生物学 Q4 BIOCHEMICAL RESEARCH METHODS

Protein expression and purification Pub Date : 2024-09-18 DOI:10.1016/j.pep.2024.106609

引用次数: 0

Abstract

The recognition and characterization of gene-encoded nitric oxide synthase (NOS) from Exiguobacterium profundum are reported in this study. A new gene was sequenced and cloned from E. profundum and heterologously expressed in E. coli for functional identification, followed by protein purification using the His-tag. The stability and activity characteristics of the recombinant NOS were evaluated using different concentrations of IPTG at various time points. A band of approximately 42 kDa was observed by SDS-PAGE. The K_m value of NOS, calculated based on the Michaelis-Menten equation was 0.59 μmol/L. Additionally, homologous sequence alignment analysis indicated that the new NOS shared 80.48 % similarity with the same protein from Bacillus subtilis and Umezawaea. The construction of the NOS expression vector and the purification of the recombinant protein provide a foundation for further functional research and inhibitor development.

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一氧化氮合成酶基因的克隆和特性鉴定及其在大肠杆菌中的表达。

本研究报告了对深海外杆菌基因编码的一氧化氮合酶（NOS）的识别和表征。研究人员对深沟外杆菌的一个新基因进行了测序和克隆，并在大肠杆菌中进行了异源表达以进行功能鉴定，随后使用 His 标记对蛋白质进行了纯化。在不同时间点使用不同浓度的 IPTG 对重组 NOS 的稳定性和活性特征进行了评估。通过 SDS-PAGE 可以观察到一条大约 42 kDa 的条带。根据 Michaelis-Menten 方程计算，NOS 的 Km 值为 0.59 μmol/L。此外，同源序列比对分析表明，新的 NOS 与枯草芽孢杆菌和梅泽藻中的相同蛋白有 80.48% 的相似性。NOS表达载体的构建和重组蛋白的纯化为进一步的功能研究和抑制剂开发奠定了基础。

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.