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.

清道夫受体是一种蛋白质超家族，通常由清道夫受体富含半胱氨酸结构域 (SRCRD) 的一个或多个重复组成，SRCRD 是一种古老且高度保守的蛋白质模块。含有多个二硫键的真核蛋白的表达和纯化一直具有挑战性。常用的表达SRRCD蛋白的表达系统主要由真核蛋白表达系统组成。在此，我们利用大肠杆菌表达系统建立了人唾液凝集素的 B 型 SRCRD 单元的高水平表达策略，然后进行重折叠和纯化过程。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过程。大肠杆菌SHuffle T7中表达的SRRCD在重折叠后显示出比大肠杆菌BL21(DE3)中表达的更好的溶解度，这表明重折叠前二硫键含量的重要性。最终使用结晶和晶体结构分析评估重折叠蛋白质的质量。由于从包涵体中重折叠的蛋白质表现出高晶体质量和重现性，因此该方法被认为是 SRCRD 蛋白质表达和纯化的可靠策略。为了进一步确认重折叠的SRRCD蛋白的结构完整性，使用坐滴蒸气扩散法对纯化的蛋白进行结晶。获得的 SRCRD 晶体衍射 X 射线分辨率为 1.47 Å。解析出的晶体结构似乎高度保守，适当形成了四个二硫键。同源 SRCRD 蛋白的表面电荷分布表明表面的负电荷区域与其钙依赖性配体识别相关。这些结果表明，大肠杆菌SHuffle T7表达的高质量SRRCD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新的选择。