He M, Liu H, Austen B
Department of Surgery, St. George's Hospital Medical School, London, UK.
DNA Cell Biol. 1994 Aug;13(8):875-82. doi: 10.1089/dna.1994.13.875.
The aim of this work was to express a eukaryotic pre-protein in Escherichia coli so that it could be obtained intact, without cleavage, by bacterial leader peptidase. To this end, cDNA coding for honeybee prepromelittin was ligated to the 3' end of genes coding for truncated forms of either Protein A or beta-galactosidase (beta-Gal) under the control of inducible promoters, with an oligonucleotide coding for the Factor Xa cleavage site at the junction between the two proteins. The Protein A fusion was expressed in good yield, and about 80% of it formed inclusion bodies. The prepromelittin section of the Protein A fusion caused some export of the intact fusion protein to the growth medium. The prepromelittin beta-Gal fusion was expressed in low yield and became associated with the E. coli cytoplasmic membrane. Its expression was toxic to E. coli. Thus, the synthesis of a full-length eukaryotic pre-protein in E. coli is best achieved when the fusion protein forms inclusion bodies.
这项工作的目的是在大肠杆菌中表达一种真核前体蛋白，以便通过细菌前导肽酶完整地获得它，而不发生切割。为此，将编码蜜蜂前原蜂毒肽的cDNA连接到在诱导型启动子控制下编码截短形式的A蛋白或β-半乳糖苷酶（β-Gal）的基因的3'端，并在两种蛋白质之间的连接处用编码因子Xa切割位点的寡核苷酸。A蛋白融合体表达量很高，其中约80%形成包涵体。A蛋白融合体的前原蜂毒肽部分导致完整的融合蛋白向生长培养基的一些输出。前原蜂毒肽β-Gal融合体表达量很低，并与大肠杆菌细胞质膜结合。它的表达对大肠杆菌有毒。因此，当融合蛋白形成包涵体时，在大肠杆菌中合成全长真核前体蛋白的效果最佳。
