Kiss R S, Kay C M, Ryan R O
Department of Biochemistry, University of Alberta, Edmonton, Alberta, T6G 2S2, Canada.
Protein Expr Purif. 1998 Apr;12(3):353-60. doi: 10.1006/prep.1997.0853.
Apolipoprotein (apo) A-I is a 28-kDa exchangeable apolipoprotein that plays a key role in lipoprotein metabolism. It is widely distributed among animal species and is rich in alpha-helical secondary structure. Unlike human apoA-I, which aggregates in the absence of lipid, chicken apoA-I is monomeric in the lipid-free state. To take advantage of this physical characteristic, a bacterial expression system for production of recombinant chicken apoA-I has been developed. The cDNA-encoding chicken apoA-I was cloned into the pET expression vector under the regulation of the lac operon and transformed into Escherichia coli. Recombinant apoA-I protein recovered from the soluble fraction of the bacterial cell pellet was purified to greater than 95% homogeneity by reversed-phase high-performance liquid chromatography. Although immunoblot analysis confirmed the identity of the overexpressed protein, its migration on denaturing polyacrylamide gel electrophoresis was slower than its natural counterpart. To determine if the vector-encoded 18 residue pelB N-terminal leader sequence was not cleaved by the bacterial leader peptidase, isolated recombinant chicken apoA-I was incubated with exogenous leader peptidase. This treatment resulted in an increased electrophoretic mobility, with migration to a position corresponding to plasma-derived chicken apoA-I. Electrospray mass spectrometry indicated a mass of 27,961 +/- 4 Da, in agreement with that predicted for natural chicken apoA-I. Far-UV circular dichroism spectroscopy indicated an alpha-helical content similar to apoA-I isolated from chicken plasma, suggesting that the protein is folded in solution. Fluorescence studies showed that the wavelength of maximum fluorescence emission of the two tryptophan residues in the protein was 331 nm, with no shift occurring following complexation with lipid. Recombinant apoA-I was shown to be functional in lipoprotein binding as well as to possess an ability to transform bilayer vesicles of dimyristoylphosphatidylcholine into discoidal complexes. This is the first report of bacterial expression of an avian apoA-I. Increased availability and the potential for site-directed mutagenesis of this protein will aid in further characterization of apoA-I and the mechanism whereby it functions in cholesterol transport.
载脂蛋白（apo）A-I是一种28 kDa的可交换载脂蛋白，在脂蛋白代谢中起关键作用。它广泛分布于动物物种中，富含α-螺旋二级结构。与在无脂质时会聚集的人apoA-I不同，鸡apoA-I在无脂质状态下是单体形式。为利用这一物理特性，已开发出用于生产重组鸡apoA-I的细菌表达系统。编码鸡apoA-I的cDNA在乳糖操纵子的调控下克隆到pET表达载体中，并转化到大肠杆菌中。从细菌细胞沉淀的可溶部分回收的重组apoA-I蛋白通过反相高效液相色谱纯化至纯度大于95%。尽管免疫印迹分析证实了过表达蛋白的身份，但其在变性聚丙烯酰胺凝胶电泳上的迁移速度比天然对应物慢。为确定载体编码的18个残基的pelB N端前导序列是否未被细菌前导肽酶切割，将分离的重组鸡apoA-I与外源前导肽酶一起孵育。这种处理导致电泳迁移率增加，迁移到与血浆来源的鸡apoA-I相对应的位置。电喷雾质谱表明其质量为27,961±4 Da，与天然鸡apoA-I的预测质量一致。远紫外圆二色光谱表明其α-螺旋含量与从鸡血浆中分离的apoA-I相似，表明该蛋白在溶液中折叠。荧光研究表明，该蛋白中两个色氨酸残基的最大荧光发射波长为331 nm，与脂质复合后未发生位移。重组apoA-I在脂蛋白结合方面具有功能，并且具有将二肉豆蔻酰磷脂酰胆碱双层囊泡转化为盘状复合物的能力。这是关于禽类apoA-I细菌表达的首次报道。该蛋白可用性的增加以及定点诱变的潜力将有助于进一步表征apoA-I及其在胆固醇转运中的作用机制。
