Ding Man-Sheng, Ma Wen-Feng, Zhang Mei-Fang, Liu Da-Tao, Guo Mei-Jin, Zhuang Ying-Ping, Chu Ju, Zhang Si-Liang, Gong Bang-Qiang
State Key Laboratory of Bioreactor Engineering, East China University of Science and Technology, Shanghai 200237, China.
Sheng Wu Gong Cheng Xue Bao. 2005 Mar;21(2):198-203.
Apolipoprotein AI (apo AI), the major protein component of human high-density lipoprotein (HDL), is a single-chain polypeptide of 243 amino acids. Several epidemiological studies have shown that the plasma concentrations of HDL has the role of reverse cholesterol transport (RCT) and inversely correlated with the incidence of coronary artery disease. Because apo AI lacks post-translational modifications, it is convenient to express human apo AI in Escherichia coli expression system. However, there is a poor stability of the mRNA and the apo AI protein in E. coli, it is difficult to express mature apo AI in recombinant bacteria, moreover, even as a fusion protein, apo AI is still sensitive to degradation and can not be cleaved efficiently from the fusion tags. In contrast, proapolipoprotein AI (proapo AI, having an additional polypeptide containing the amino acids Arg-His-Phe-Trp-Gln-Gln at the amino-teminal of the mature protein) proved stable and undegraded in Escherichia coli, and therefore, in this research, an expression system of E. coli including a plasmid of P(R)P(L) tandem promoter was adapted to produce proapo AI. Furthermore, site-directed mutagenesis of the proapo AI cDNA was performed to generate a Clu8Asp mutation in the amino-terminal sequence of proapo AI which created an acid labile Asp-Pro peptide bond between amino acid 8 and 9, and permitted specific chemical cleavage to remove pro-peptide. After inducing with a shift of temperature, yields of recombinant proapo AI achieved about 40% of total cell protein and the recombinant proapo AI expressed proved as a form of inclusion body in cells, so protein need to renature. First of all, the protein was dissolved in buffer with denaturant, and renaturation was carried out on a hydrophobic interaction column (Phenyl Sepharose), ion-exchange chromatography and gel-filtration chromatography were then used to further purify the protein. The purified recombinant apo AI was detected by a set of tests including Western-blotting, Circular dichroism spectra and lipid-binding test, the results shown that recombinant apo AI has similar structural and lipid-binding properties identical to those of native plasma apo AI, which facilitates further research and application.
载脂蛋白AI（apo AI）是人类高密度脂蛋白（HDL）的主要蛋白质成分，是一种由243个氨基酸组成的单链多肽。多项流行病学研究表明，HDL的血浆浓度具有逆向胆固醇转运（RCT）的作用，且与冠状动脉疾病的发病率呈负相关。由于apo AI缺乏翻译后修饰，因此在大肠杆菌表达系统中表达人apo AI较为方便。然而，apo AI的mRNA和蛋白质在大肠杆菌中的稳定性较差，在重组菌中难以表达成熟的apo AI，而且，即使作为融合蛋白，apo AI仍然对降解敏感，无法从融合标签上有效切割下来。相比之下，前载脂蛋白AI（proapo AI，在成熟蛋白的氨基末端有一个额外的包含氨基酸Arg-His-Phe-Trp-Gln-Gln的多肽）在大肠杆菌中被证明是稳定且未降解的，因此，在本研究中，采用了一种包含P(R)P(L)串联启动子质粒的大肠杆菌表达系统来生产proapo AI。此外，对proapo AI cDNA进行了定点诱变，在proapo AI的氨基末端序列中产生了一个Clu8Asp突变，该突变在氨基酸8和9之间形成了一个酸不稳定的Asp-Pro肽键，并允许进行特异性化学切割以去除前肽。经温度转换诱导后，重组proapo AI的产量达到总细胞蛋白的约40%，且表达的重组proapo AI在细胞中以包涵体的形式存在，因此蛋白质需要复性。首先，将蛋白质溶解在含有变性剂的缓冲液中，然后在疏水相互作用柱（苯基琼脂糖）上进行复性，接着使用离子交换色谱和凝胶过滤色谱进一步纯化蛋白质。通过包括蛋白质免疫印迹、圆二色光谱和脂质结合试验在内的一系列测试对纯化的重组apo AI进行检测，结果表明重组apo AI具有与天然血浆apo AI相似的结构和脂质结合特性，这有利于进一步的研究和应用。
