title: Expression, purification, and refolding of active human and mouse secreted group IIE phospholipase A₂.
abstract: Secreted phospholipase A₂s form a large family of proteins involved in diverse biological and pathophysiological processes. Group IIE secreted phospholipase A₂ (sPLA₂-IIE) is one of the latest discovered members of this family. Previous studies revealed that the expression profile of sPLA₂-IIE was restricted to a few tissue types including brain, heart, lung and placenta compared to the broad expression profile of other isoforms. Accumulating evidence suggests that sPLA₂-IIE might play a pivotal role in the progression of inflammatory processes. However, functional study of sPLA₂-IIE was hindered by the low yield of soluble expressed protein. In this study, we have expressed human and mouse sPLA₂-IIE in Escherichia coli in the form of inclusion bodies. The inclusion bodies were dissolved, purified and refolded in a step-wise dialysis approach and further purified. We obtained soluble and active proteins for human and mouse sPLA₂-IIE with a final yield of 1.1 and 1.2 mg/500 mL bacterial culture, respectively. The refolded sPLA₂-IIEs exhibited similar calcium and pH dependence of their enzymatic activity with those expressed in COS cells. This protein expression and purification protocol will facilitate the further structural and functional studies of human and mouse sPLA₂-IIEs.
authors: State Key Laboratory of Respiratory Disease, Guangzhou Institutes of Biomedicine and Health, Chinese Academy of Sciences, People's Republic of China.
authors: Yujie
authors: Y
authors: Zhang
authors: Tingting
authors: T
authors: Xu
authors: Qiujia
authors: Q
authors: Chen
authors: Bing
authors: B
authors: Wang
authors: Jinsong
authors: J
authors: Liu