Fisher C A, Wang J, Francis G A, Sykes B D, Kay C M, Ryan R O
Lipid and Lipoprotein Research Group, University of Alberta, Edmonton, Canada.
Biochem Cell Biol. 1997;75(1):45-53.
The nucleotide sequence encoding the N-terminal domain (residues 1-183) of human apolipoprotein E3 (apoE3) was cloned into the pET expression vector and introduced into Escherichia coli. Induction of protein expression with isopropyl beta-D-thiogalactopyranoside resulted in production of recombinant apoE3(1-183). Immunoblot analysis revealed that recombinant protein was present in both the cell pellet and cell culture supernatant. Analysis revealed that a significant portion of the rApoE3(1-183) in the cell pellet still possessed the bacterial N-terminal pel B leader sequence, encoded by plasmid DNA directly upstream of the apoE3(1-183) coding sequence. By contrast, this hydrophobic leader sequence had been removed from recombinant protein specifically accumulating in the culture medium. This behavior is novel for bacterial expression of apolipoprotein E and its truncated variants and permits efficient overexpression of the recombinant protein (> 100 mg/L cell culture). Recombinant apoE3(1-183) was isolated by a combination of heparin-Sepharose chromatography and reverse-phase HPLC. Electrospray mass spectrometry provided a mass of 21 191 daltons, corresponding directly to that expected from the known sequence. Circular dichroism spectroscopy revealed that the recombinant protein possesses significant amounts of alpha-helical secondary structure. The lipid binding ability of rApoE3(1-183) was evaluated using an in vitro lipoprotein binding assay. It was observed that recombinant apoE3(1-183) protected human low density lipoprotein (LDL) from lipid accumulation induced particle aggregation, indicating that it is capable of associating with lipoprotein surfaces. In addition, rApoE3(1-183) forms disk complexes with the model phospholipid dimyristoylphosphatidylcholine. In competition experiments, it was observed that rApoE3(1-183) phospholipid disks compete with 125I-LDL for binding to the apoB/E receptor on human skin fibroblasts to an extent similar to that observed for intact rApoE3. Taken together, these data show that recombinant apoE3(1-183) is fully functional as an apolipoprotein and receptor ligand. Given the high expression level and its known existence as a monomer in solution, we evaluated the potential for application of NMR spectroscopy to study the structure-function relationship of rApoE3(1-183). Bacteria were cultured in media supplemented with 15NH4Cl or [15N]glycine and the isotopically labeled recombinant apoE3(1-183) was analyzed by heteronuclear single quantum correlation NMR spectroscopy. The data revealed that rApoE3(1-183) is an excellent candidate for solution structure studies by NMR, including conformational adaptations associated with lipid association.
编码人载脂蛋白E3（apoE3）N端结构域（第1至183位氨基酸残基）的核苷酸序列被克隆到pET表达载体中，并导入大肠杆菌。用异丙基β-D-硫代半乳糖苷诱导蛋白表达，产生了重组apoE3(1-183)。免疫印迹分析表明，重组蛋白存在于细胞沉淀和细胞培养上清液中。分析发现，细胞沉淀中的大部分rApoE3(1-183)仍具有细菌N端pelB前导序列，该序列由apoE3(1-183)编码序列直接上游的质粒DNA编码。相比之下，这种疏水前导序列已从特异性积累在培养基中的重组蛋白中去除。这种行为对于载脂蛋白E及其截短变体的细菌表达来说是新颖的，并且能够高效过量表达重组蛋白（>100mg/L细胞培养物）。通过肝素-琼脂糖层析和反相高效液相色谱相结合的方法分离重组apoE3(1-183)。电喷雾质谱分析得到的分子量为21191道尔顿，与已知序列预期的分子量直接对应。圆二色光谱表明，重组蛋白具有大量的α-螺旋二级结构。使用体外脂蛋白结合试验评估rApoE3(1-183)的脂质结合能力。观察到重组apoE3(1-183)可保护人低密度脂蛋白（LDL）免受脂质积累诱导的颗粒聚集，表明它能够与脂蛋白表面结合。此外，rApoE3(1-183)与模型磷脂二肉豆蔻酰磷脂酰胆碱形成盘状复合物。在竞争实验中，观察到rApoE3(1-183)磷脂盘与125I-LDL竞争结合人皮肤成纤维细胞上的apoB/E受体，其竞争程度与完整的rApoE3相似。综上所述，这些数据表明重组apoE3(1-183)作为载脂蛋白和受体配体具有完全的功能。鉴于其高表达水平以及已知在溶液中以单体形式存在，我们评估了应用核磁共振波谱研究rApoE3(1-183)结构-功能关系的潜力。细菌在补充有15NH4Cl或[15N]甘氨酸的培养基中培养，并用异核单量子相关核磁共振波谱分析同位素标记的重组apoE3(1-183)。数据表明，rApoE3(1-183)是通过核磁共振进行溶液结构研究的极佳候选物，包括与脂质结合相关的构象适应性研究。
