Nagai K, Thøgersen H C
Nature. 1984;309(5971):810-2. doi: 10.1038/309810a0.
High-level expression of many eukaryotic genes has proved difficult to achieve even when a strong promoter and the ribosome binding sequence from highly expressed Escherichia coli genes have been placed in front of the coding sequences. To overcome this problem, many eukaryotic proteins have been efficiently produced as hybrids after fusion of their genes with a coding sequence of E. coli genes. However, such hybrid proteins are not suitable for functional studies or clinical use unless the authentic protein sequence can be released by specific cleavage. Here, we have inserted the sequence Ile-Glu-Gly-Arg between the 31 amino-terminal residues of lambda cII protein and Val 1 of human beta-globin, and produced this hybrid in high yield in E. coli. We then cleaved the hybrid specifically at the single arginine, using blood coagulation factor Xa and thus liberated the authentic beta-globin chain. As factor Xa is specific for the tetrapeptide Ile-Glu-Gly-Arg, which is rare in protein sequences, our expression/cleavage system is applicable to the efficient production of many eukaryotic proteins.
即使在编码序列前放置了强启动子和来自高表达大肠杆菌基因的核糖体结合序列，许多真核基因的高水平表达仍难以实现。为克服这一问题，许多真核蛋白在其基因与大肠杆菌基因编码序列融合后作为杂种高效产生。然而，除非能通过特异性切割释放出真实的蛋白序列，否则此类杂种蛋白不适用于功能研究或临床应用。在此，我们在λ cII蛋白的31个氨基末端残基与人β-珠蛋白的Val 1之间插入了序列Ile-Glu-Gly-Arg，并在大肠杆菌中高产表达了这种杂种蛋白。然后，我们使用凝血因子Xa在单个精氨酸处特异性切割该杂种蛋白，从而释放出真实的β-珠蛋白链。由于因子Xa对四肽Ile-Glu-Gly-Arg具有特异性，而该四肽在蛋白质序列中很少见，我们的表达/切割系统适用于许多真核蛋白的高效生产。
