摘要

用于工业生产的表达载体应该是稳定的并且允许严格控制蛋白质合成。这对于确保质粒向子细胞的传递是必要的，以便获得能够合成大量靶蛋白的稳定群体。高拷贝数质粒pAE以前用于实验室规模生产重组人粒细胞集落刺激因子（rhG-CSF）和曼氏血吸虫脂肪酸结合蛋白（rSm14），但它对大型不稳定规模生产。因此，在这里我们评估了一种来自pAE，pAR-KanI的新表达载体，它结合了两种质粒复制策略：作为pAE的高拷贝质粒pUC复制起点和来自pSC101的par基因座序列，这是典型的低拷贝质粒，用于在大肠杆菌中产生rhG-CSF和rSm14。携带这些构建体的克隆在两种复合培养基（2YT和自诱导）中培养，并且在诱导之前和之后两者都产生高于95％的抗性菌落，有或没有抗生素。在2YT培养基中，我们分别获得244μg/ mL的rSm14,181μg/ mL和392μg/ mL的rhG-CSF，分别含有和不含葡萄糖。在不含抗生素的自诱导培养基中，获得147μg/ mL的rSm14和162μg/ mL的rhG-CSF。新载体对于在大肠杆菌中的复杂培养基中生产两种重组蛋白具有高稳定性，甚至在不存在抗生素的情况下，使得pAR-KanI成为重组蛋白的工业生产的有希望的载体。

Expression vectors for industrial production should be stable and allow tight control of protein synthesis. This is necessary to ensure plasmid transmission to daughter cells in order to achieve a stable population capable of synthesizing high amounts of the target protein. A high-copy-number plasmid, pAE, was previously used for laboratory-scale production of recombinant human granulocyte colony-stimulating factor (rhG-CSF) and the Schistosoma mansoni fatty acid binding protein (rSm14), but it was unstable for large-scale production. Therefore, here we evaluated a new expression vector derived from pAE, pAR-KanI, which combines two plasmid replication strategies: a high-copy plasmid pUC origin of replication as pAE, and a par locus sequence derived from pSC101, which is typical of low copy plasmids, for rhG-CSF and rSm14 production in Escherichia coli. Clones bearing these constructs were cultivated in two complex media (2YT and auto-induction) and both yielded higher-than-95% resistant colonies, before and after induction, either with or without antibiotics. In 2YT medium, we obtained 244 μg/mL of rSm14, 181 μg/mL and 392 μg/mL for rhG-CSF, with and without glucose, respectively. In auto-induction medium without antibiotics, 147 μg/mL of rSm14 and 162 μg/mL of rhG-CSF were obtained. The new vector presented high stability for the production of both recombinant proteins in complex media in Escherichia coli, even in the absence of antibiotics, making the pAR-KanI a promising vector for industrial production of recombinant proteins.

https://www.ncbi.nlm.nih.gov/pubmed/30193910