PROTEIN SECRETION CONTROLLED BY A SYNTHETIC GENE IN ESCHERICHIA-COLI
PROTEIN SECRETION CONTROLLED BY A SYNTHETIC GENE IN ESCHERICHIA-COLI
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DOI：
10.1093/protein/2.6.473
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发表时间：
1989-03-01
期刊：
影响因子：
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通讯作者：
MASSON, JM
中科院分区：
文献类型：
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作者：
BLANCHINROLAND, S;MASSON, JM
大肠杆菌不能在生长介质中分泌蛋白质是其在基因工程中应用的主要缺陷之一。为了在大肠杆菌中诱导分泌同源或异源蛋白，在lac启动子的控制下，合成并克隆了一个与pColE1的kIL多肽同源的基因。通过分析地衣芽孢杆菌的R-TEM1β-内酰胺酶和α-淀粉酶两种蛋白质的产生和分泌，分析了该合成基因促进分泌的效率。后一种蛋白在大肠杆菌中通过其基因在与kIL基因相同的质粒上或在不同的质粒上表达。KIL基因诱导的主要作用是分泌蛋白的过量生产。当高水平表达时，KIL基因促进所有周质蛋白的过量生产和培养基中β-内酰胺酶或α-淀粉酶的总分泌。这种分泌是半选择性的，因为大多数周质蛋白是不分泌的。KIL多肽通过两个步骤诱导同源或异源蛋白的分泌，首先作用于细胞膜，然后通透外膜。该系统目前正在发酵罐规模上进行测试，是第一个使用合成基因将新特性改造成细菌菌株的例子。
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 protein 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.