大肠杆菌中表达的人唾液凝集素清道夫受体富半胱氨酸结构域的重折叠、结晶和晶体结构分析。

IF 1.9 4区 生物学 Q4 BIOCHEMISTRY & MOLECULAR BIOLOGY The Protein Journal Pub Date : 2024-01-24 DOI:10.1007/s10930-023-10173-x
Changyu Zhang, Peng Lu, Sibo Wei, Chaoyue Hu, Mitsuko Miyoshi, Ken Okamoto, Hideaki Itoh, Suguru Okuda, Michio Suzuki, Hiroshi Kawakami, Koji Nagata
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引用次数: 0

摘要

清道夫受体是一个蛋白质超家族,通常由一个或多个重复的清道夫受体富半胱氨酸结构域(SCRD)组成,这是一个古老而高度保守的蛋白质模块。表达和纯化含有多个二硫键的真核蛋白质一直是一项挑战。常用于表达 SRCRD 蛋白的表达系统主要包括真核蛋白质表达系统。在此,我们利用大肠杆菌表达系统建立了一种从人类唾液凝集素中提取的 B 型 SRCRD 单元的高水平表达策略,随后进行了重折叠和纯化过程。使用 pET-32a 载体在大肠杆菌中表达未标记的重组 SRCRD,然后使用 GSH/GSSG 氧化还原系统进行重折叠。在大肠杆菌 SHuffle T7 中表达的 SRCRD 在重折叠后比在大肠杆菌 BL21(DE3) 中表达的 SRCRD 表现出更好的溶解性,这表明重折叠前二硫键含量的重要性。最后,利用结晶和晶体结构分析评估了重折叠蛋白质的质量。由于从包涵体中重折的蛋白质具有较高的晶体质量和重现性,因此这种方法被认为是 SRCRD 蛋白表达和纯化的可靠策略。为了进一步确认重折叠 SRCRD 蛋白的结构完整性,纯化后的蛋白采用坐滴蒸气扩散法进行结晶。得到的 SRCRD 晶体的 X 射线衍射分辨率为 1.47 Å。同源 SRCRD 蛋白的表面电荷分布表明,其表面的负电荷区域与钙依赖性配体识别有关。这些结果表明,用大肠杆菌 SHuffle T7 表达的高质量 SRCRD 蛋白可以成功折叠和纯化,为清道夫受体超家族成员的表达提供了新的选择。
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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Refolding, Crystallization, and Crystal Structure Analysis of a Scavenger Receptor Cysteine-Rich Domain of Human Salivary Agglutinin Expressed in Escherichia coli

Scavenger receptors are a protein superfamily that typically consists of one or more repeats of the scavenger receptor cysteine-rich structural domain (SRCRD), which is an ancient and highly conserved protein module. The expression and purification of eukaryotic proteins containing multiple disulfide bonds has always been challenging. The expression systems that are commonly used to express SRCRD proteins mainly consist of eukaryotic protein expression systems. Herein, we established a high-level expression strategy of a Type B SRCRD unit from human salivary agglutinin using the Escherichia coli expression system, followed by a refolding and purification process. The untagged recombinant SRCRD was expressed in E. coli using the pET-32a vector, which was followed by a refolding process using the GSH/GSSG redox system. The SRCRD expressed in E. coli SHuffle T7 showed better solubility after refolding than that expressed in E. coli BL21(DE3), suggesting the importance of the disulfide bond content prior to refolding. The quality of the refolded protein was finally assessed using crystallization and crystal structure analysis. As proteins refolded from inclusion bodies exhibit a high crystal quality and reproducibility, this method is considered a reliable strategy for SRCRD protein expression and purification. To further confirm the structural integrity of the refolded SRCRD protein, the purified protein was subjected to crystallization using sitting-drop vapor diffusion method. The obtained crystals of SRCRD diffracted X-rays to a resolution of 1.47 Å. The solved crystal structure appeared to be highly conserved, with four disulfide bonds appropriately formed. The surface charge distribution of homologous SRCRD proteins indicates that the negatively charged region at the surface is associated with their calcium-dependent ligand recognition. These results suggest that a high-quality SRCRD protein expressed by E. coli SHuffle T7 can be successfully folded and purified, providing new options for the expression of members of the scavenger receptor superfamily.

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来源期刊
The Protein Journal
The Protein Journal 生物-生化与分子生物学
CiteScore
5.20
自引率
0.00%
发文量
57
审稿时长
12 months
期刊介绍: The Protein Journal (formerly the Journal of Protein Chemistry) publishes original research work on all aspects of proteins and peptides. These include studies concerned with covalent or three-dimensional structure determination (X-ray, NMR, cryoEM, EPR/ESR, optical methods, etc.), computational aspects of protein structure and function, protein folding and misfolding, assembly, genetics, evolution, proteomics, molecular biology, protein engineering, protein nanotechnology, protein purification and analysis and peptide synthesis, as well as the elucidation and interpretation of the molecular bases of biological activities of proteins and peptides. We accept original research papers, reviews, mini-reviews, hypotheses, opinion papers, and letters to the editor.
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