Apolipoprotein A-I (ApoA-I) is the major protein constituent of high-density lipoprotein particles, and as such is involved in cholesterol transport and activation of LCAT (the lecithin:cholesterol acyltransferase). It may also form amyloidal deposits in the body, showing the multifaceted interactions of ApoA-I. In order to facilitate the study of ApoA-I in various systems, we have developed a protocol based on recombinant expression in E. coli. ApoA-I is protected from degradation by driving its expression to inclusion bodies using a tag: the EDDIE mutant of Npro autoprotease from classical swine fever virus. Upon refolding, EDDIE will cleave itself off from the target protein. The result is a tag-free ApoA-I, with its N-terminus intact. ApoA-I was then purified using a five-step procedure composed of anion exchange chromatography, immobilized metal ion affinity chromatography, hydrophobic interaction chromatography, boiling and size exclusion chromatography. This led to protein of high purity as confirmed with SDS-PAGE and mass spectrometry. The purified ApoA-I formed discoidal objects in the presence of zwitterionic phospholipid DMPC, showing its retained function of interacting with lipids. The protocol was also tested by expression and purification of two ApoA-I mutants, both of which could be purified in the same manner as the wildtype, showing the robustness of the protocol.

载脂蛋白 AI (ApoA-I) 是高密度脂蛋白颗粒的主要蛋白质成分，因此参与胆固醇转运和 LCAT（卵磷脂：胆固醇酰基转移酶）的激活。它也可能在体内形成淀粉样沉积物，显示出 ApoA-I 的多方面相互作用。为了促进 ApoA-I 在各种系统中的研究，我们开发了一种基于大肠杆菌中重组表达的方案. ApoA-I 通过使用标签驱动其表达至包涵体来防止降解：来自经典猪瘟病毒的 Npro 自蛋白酶的 EDDIE 突变体。重新折叠后，EDDIE 会将自身从靶蛋白上切割下来。结果是一个无标签的 ApoA-I，其 N 末端完好无损。然后使用由阴离子交换色谱、固定化金属离子亲和色谱、疏水相互作用色谱、沸腾和尺寸排阻色谱组成的五步程序纯化 ApoA-I。如 SDS-PAGE 和质谱所证实的，这导致了高纯度的蛋白质。纯化的ApoA-I在两性离子磷脂DMPC存在下形成盘状物体，显示其保留与脂质相互作用的功能。该协议还通过两个 ApoA-I 突变体的表达和纯化进行了测试，