Blanchin-Roland S, Masson J M
INSA, Département de Génie Biochimique et alimentaire, Centre de Transfert en Biotechnologie-Microbiologie, Toulouse, France.
Protein Eng. 1989 Mar;2(6):473-80. doi: 10.1093/protein/2.6.473.
The inability of Escherichia coli to secrete proteins in growth medium is one of the major drawbacks in its use in genetic engineering. A synthetic gene, homologous to the one coding for the kil peptide of pColE1, was made and cloned under the control of the lac promoter, in order to obtain the inducible secretion of homologous or heterologous proteins by E. coli. The efficiency of this synthetic gene to promote secretion was assayed by analysing the production and secretion of two proteins, the R-TEM1 beta-lactamase, and the alpha-amylase from Bacillus licheniformis. This latter protein was expressed in E. coli from its gene either on the same plasmid as the kil gene or on a different plasmid. The primary effect of the induction of the kil gene is the overproduction of the secreted proteins. When expressed at a high level, the kil gene promotes the overproduction of all periplasmic proteins and the total secretion in the culture medium of both the beta-lactamase or the alpha-amylase. This secretion is semi-selective for most periplasmic proteins are not secreted. The kil peptide induces the secretion of homologous or heterologous proteins in two steps, first acting on the cytoplasmic membrane, then permeabilizing the outer membrane. This system, which is now being assayed at the fermentor scale, is the first example of using a synthetic gene to engineer a new property into a bacterial strain.
大肠杆菌无法在生长培养基中分泌蛋白质是其在基因工程应用中的主要缺陷之一。构建了一个与编码pColE1 kil肽的基因同源的合成基因，并将其克隆到乳糖启动子的控制下，以便通过大肠杆菌实现同源或异源蛋白质的诱导分泌。通过分析两种蛋白质（R-TEM1β-内酰胺酶和地衣芽孢杆菌的α-淀粉酶）的产生和分泌来检测该合成基因促进分泌的效率。后一种蛋白质在大肠杆菌中由其基因表达，该基因要么与kil基因在同一质粒上，要么在不同质粒上。kil基因诱导的主要作用是分泌蛋白的过量产生。当高水平表达时，kil基因促进所有周质蛋白的过量产生以及β-内酰胺酶或α-淀粉酶在培养基中的总分泌。这种分泌对大多数周质蛋白不分泌具有半选择性。kil肽分两步诱导同源或异源蛋白质的分泌，首先作用于细胞质膜，然后使外膜通透化。这个目前正在发酵罐规模进行检测的系统，是利用合成基因将新特性引入细菌菌株的首个例子。
