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的基因引入大肠杆菌等微生物中，并获得大量的基因产物。在本项目中，我们的目标是通过控制表达的重组蛋白的性质形成包涵体，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。我们以酵母线粒体MIF4p作为模型重组蛋白，其在大肠杆菌细胞中的表达效率较低。我们用SynA1、SynA2、SynB1、SynB2、SynC（酵母细胞色素氧化酶亚基IV的衍生物）和T7TAG等多肽替换MIF4p的前置序列，并制作相应的融合基因。然后将融合基因导入大肠杆菌细胞，诱导其过表达。采用免疫印迹法评估融合蛋白的表达水平，并与成熟的无序列MIF4p进行比较。以T7TAG和SynB2标记的MIF4p比成熟的MIF4p表达水平更高。在包涵体中发现了Syn B2-MIF4p融合蛋白，而在可溶性部分中发现了T7TAG-MIF4p融合蛋白。这些肽（SynB2和T7TAG）将提供一个有用的起点，进一步优化它们的能力，以提高附着蛋白在大肠杆菌细胞中的表达水平。

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.