用于核磁共振研究的淀粉样前体蛋白跨膜区域的表达、纯化和胶束重构

Expression, purification and micelle reconstruction of the transmembrane domain of the human amyloid precursor protein for NMR studies

摘要:

淀粉样前体蛋白(amyloid precursor protein, APP)被多次酶切后生成β-淀粉样蛋白(amyloid-β peptide, Aβ)，其聚合物的毒性作用会引发阿尔茨海默病(Alzheimer’s disease, AD)。其中，APP蛋白的跨膜区域(transmembrane domain of amyloid precursor protein, APPTM)与γ-分泌酶的非特异性切割作用是生成Aβ的关键步骤，在生理条件下重构APPTM对于探究其与γ-分泌酶的相互作用以及AD药物研发具有重要作用。然而，现有的重组APPTM制备方法存在制备效率和产量低等缺点，限制了APPTM的稳定大规模制备。本研究以大肠杆菌(Escherichia coli)为宿主，使用pMM-LR6载体对APPTM进行融合表达。包涵体蛋白经盐酸胍提取后，依次使用Ni-NTA亲和层析、溴化氰切割融合标签和反相高效液相色谱(reverse phase high performance liquid chromatography, RP-HPLC)，得到了高纯度和高产量的同位素标记的APPTM。进一步将APPTM重构到十二烷基磷酰胆碱(dodecylphosphocholine, DPC)胶束环境中，采集得到了均一、高质量的¹⁵N-¹H异核单量子关系(heteronuclear singular quantum correlation, HSQC)二维谱。本研究成功建立了一种高效且可靠的用于表达、纯化和体外重构APPTM的方法，为后续在更天然的模拟膜环境，如脂碟(bicelle)和纳米碟(nanodisc)中探究APPTM及其复合物的结构和功能奠定了良好的基础。

Abstract:

The multiple-step cleavage of amyloid precursor protein (APP) generates amyloid-β peptides (Aβ), highly toxic molecules causing Alzheimer's disease (AD). The nonspecific cleavage between the transmembrane region of APP (APPTM) and γ-secretase is the key step of Aβ generation. Reconstituting APPTM under physiologically-relevant conditions is crucial to investigate how it interacts with γ-secretase and for future AD drug discovery. Although producing recombinant APPTM was reported before, the large scale purification was hindered by the use of biological protease in the presence of membrane protein. Here, we expressed recombinant APPTM in Escherichia coli using the pMM-LR6 vector and recovered the fusion protein from inclusion bodies. By combining Ni-NTA chromatography, cyanogen bromide cleavage, and reverse phase high performance liquid chromatography (RP-HPLC), isotopically-labeled APPTM was obtained in high yield and high purity. The reconstitution of APPTM into dodecylphosphocholine (DPC) micelle generated mono dispersed 2D ¹⁵N-¹H HSQC spectra in high quality. We successfully established an efficient and reliable method for the expression, purification and reconstruction of APPTM, which may facilitate future investigation of APPTM and its complex in more native like membrane mimetics such as bicelle and nanodiscs.