4.2 Article

GFP fusion promotes the soluble and active expression of a pea actin isoform (PEAc1) in Escherichia coli

期刊

PREPARATIVE BIOCHEMISTRY & BIOTECHNOLOGY
卷 53, 期 5, 页码 557-564

出版社

TAYLOR & FRANCIS INC
DOI: 10.1080/10826068.2022.2114011

关键词

GFP fusion; pea; plant actin; polymerization; soluble expression

向作者/读者索取更多资源

It was found in this study that the soluble expression of a pea actin isoform (PEAc1) in E. coli was facilitated by a GFP fusion tag. The addition of a GFP fusion tag improved the solubility and activity of PEAc1, making it more similar to muscle actin. This enhancement is likely due to the correct folding of PEAc1 mediated by GFP fusion.
In the present study, we report that a GFP fusion tag facilitated the soluble expression of a pea actin isoform (PEAc1) in E. coli. To investigate the influence of a GFP fusion tag on PEAc1 structure and activity, PEAc1, His-tagged PEAc1 (His-PEAc1), His-tagged GFP (His-GFP), and His-tagged PEAc1 fusion with GFP (His-PEAc1-GFP) were expressed in E. coli. SDS-PAGE and western blot analyses reveal that the solubility of His-PEAc1-GFP was higher than that of PEAc1 and His-PEAc1. The His-PEAc1-GFP and His-GFP fusion proteins were purified from the supernatant of cell homogenate on a Ni-affinity column, and PEAc1 and His-PEAc1 were purified from inclusion bodies. CD spectrum analysis of the four purified proteins indicated that the proportion of alpha-helix and beta-sheet in PEAc1 was closest to the predicted data in His-PEAc1-GFP (compared with His-PEAc1 or PEAc1). In addition, the actin-associated activities of His-PEAc1-GFP, including polymerization to microfilaments under specific ionic conditions and DNase I inhibition by monomers, were more similar to those of muscle actin (compared with PEAc1 and His-PEAc1). These improvements in PEAc1 solubility and activity are likely the result of correct PEAc1 folding mediated by GFP fusion.

作者

我是这篇论文的作者
点击您的名字以认领此论文并将其添加到您的个人资料中。

评论

主要评分

4.2
评分不足

次要评分

新颖性
-
重要性
-
科学严谨性
-
评价这篇论文

推荐

暂无数据
暂无数据