High-level expression of recombinant proteins by controling the formation of the inclusion body

重组DNA技术的最新进展使我们能够将外源有用蛋白质的基因导入大肠杆菌等微生物中，并大量获得其基因产物。本课题以酵母线粒体MIF 4p为模型蛋白，在大肠杆菌中表达效率较低，通过控制表达的重组蛋白形成包涵体的性质，提高重组蛋白的表达效率，抑制其可能的细胞毒性，使其纯化更加可行。我们将MIF 4p的前序列分别用SynA 1、SynA 2、SynB 1、SynB 2、SynC（酵母细胞色素氧化酶亚基IV前序列的衍生物）和T7 TAG等肽段进行置换，并制备了相应的融合基因。然后我们将融合基因导入大肠杆菌细胞并诱导其过表达。通过免疫印迹法评估融合蛋白的表达水平，并与成熟的、无前序列的MIF 4p的表达水平进行比较。用T7 TAG和SynB 2标记的MIF 4p显示出比成熟MIF 4p更高的表达水平。在包涵体中发现Syn B2-MIF 4p融合蛋白，而在可溶性级分中回收T7 TAG-MIF 4p融合蛋白。这些肽（SynB 2和T7 TAG）将提供一个有用的起点，以进一步优化其提高大肠杆菌细胞中附着蛋白表达水平的能力。

Recent progress in recombinant DNA technology has allowed us to introduce genes for foreign useful proteins into microorganisms such as E.coli and to obtain their gene products in large amounts. In the present project, we aimed at, by controlling the properties of the expressed recombinant proteins to form inclusion bodies, increasing the expression efficiency of the recombinant proteins, suppressing their possible cell toxicity, and making their purification more feasible.We used yeast mitochondrial MIF4p as a model recombinant protein with low expression efficiency in E.coli cells. We replaced the presequence of MIF4p with various peptides including SynA1, SynA2, SynB1, SynB2, SynC (derivatives of the preseuqnece of yeast cytochrome oxidase subunit IV) and T7TAG,and made corresponding fusion genes. We then introduced the fusion genes into E.coli cells and induced their overexpression. The expression levels of the fusion proteins were assessed by *mmunoblotting and compared with that of the mature, presequence-less MIF4p. MIF4p tagged with T7TAG and with SynB2 showed higher expression levels than the mature MIF4p. The Syn B2-MIF4p fusion protein was found in an inclusion body while the T7TAG-MIF4p fusion protein was recovered in a soluble fraction. These peptides (SynB2 and T7TAG) will offer a useful starting point to further optimize their abilities to increase expression levels of the attached proteins in E.coli cells.