Regulating the T7 RNA polymerase expression in E. coli BL21 (DE3) to provide more host options for recombinant protein production.
Regulating the T7 RNA polymerase expression in E. coli BL21 (DE3) to provide more host options for recombinant protein production.
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调节大肠杆菌 BL21 (DE3) 中 T7 RNA 聚合酶的表达，为重组蛋白生产提供更多宿主选择
DOI：
10.1186/s12934-021-01680-6
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发表时间：
2021-09-26
影响因子：
6.4
通讯作者：
Sun XM
中科院分区：
文献类型：
--
作者：
Du F;Liu YQ;Xu YS;Li ZJ;Wang YZ;Zhang ZX;Sun XM
大肠埃希菌是原核表达系统中应用最广泛的重组蛋白生产菌。在BL21(DE3)中，编码T7 RNA聚合酶(T7 RNAP)的基因受强lacUV5启动子(PlacUV5)的控制，在一定的生长条件下，该启动子比野生型lac启动子(PlacWT)更具泄漏性和活性。这些特性不利于生产具有毒性或生长负担的重组蛋白。一方面，T7RNAP的泄漏表达导致非诱导期靶蛋白的快速产生，从而从细胞生长中吸取资源。此外，在非诱导期或诱导期，T7 RNAP产物的高表达会导致难以表达的蛋白质的大量产生，这都可能导致表达载体的丢失或表达基因的突变。因此，应开发更多表达T7RNAP的不同表达水平的BL21(DE3)衍生变异株。因此，我们分别用阿拉伯糖启动子(ParaBAD)、鼠李糖启动子(PrhaBAD)、四环素启动子(PTET)等其他诱导型启动子替换PlacUV5，以期通过调节T7 RNAP的转录水平和泄漏水平来优化重组蛋白的生产。与BL21(DE3)相比，所构建的工程菌对诱导剂具有较高的敏感性，其中鼠李糖和四环素启动子的渗漏能力最低。在葡萄糖脱氢酶的生产中，工程菌BL21(DE3：：Ara)的生物量、细胞存活率和外源蛋白的表达水平均高于BL21(DE3)。此外，这些工程菌还成功地用于提高膜蛋白的产量，包括大肠杆菌胞嘧啶转运蛋白(CODB)、大肠杆菌膜蛋白插入酶/折叠酶(YIDC)和大肠杆菌F-ATPase亚单位b(ECB)。本文构建的工程菌为重组蛋白的生产提供了更多的宿主选择。
Escherichia coli is the most widely used bacterium in prokaryotic expression system for the production of recombinant proteins. In BL21 (DE3), the gene encoding the T7 RNA polymerase (T7 RNAP) is under control of the strong lacUV5 promoter (PlacUV5), which is leakier and more active than wild-type lac promoter (PlacWT) under certain growth conditions. These characteristics are not advantageous for the production of those recombinant proteins with toxic or growth-burdened. On the one hand, leakage expression of T7 RNAP leads to rapid production of target proteins under non-inducing period, which sucks resources away from cellular growth. Moreover, in non-inducing or inducing period, high expression of T7 RNAP production leads to the high-production of hard-to-express proteins, which may all lead to loss of the expression plasmid or the occurrence of mutations in the expressed gene. Therefore, more BL21 (DE3)-derived variant strains with rigorous expression and different expression level of T7 RNAP should be developed. Hence, we replaced PlacUV5 with other inducible promoters respectively, including arabinose promoter (ParaBAD), rhamnose promoter (PrhaBAD), tetracycline promoter (Ptet), in order to optimize the production of recombinant protein by regulating the transcription level and the leakage level of T7 RNAP. Compared with BL21 (DE3), the constructed engineered strains had higher sensitivity to inducers, among which rhamnose and tetracycline promoters had the lowest leakage ability. In the production of glucose dehydrogenase (GDH), a protein that causes host autolysis, the engineered strain BL21 (DE3::ara) exhibited higher biomass, cell survival rate and foreign protein expression level than that of BL21 (DE3). In addition, these engineered strains had been successfully applied to improve the production of membrane proteins, including E. coli cytosine transporter protein (CodB), the E. coli membrane protein insertase/foldase (YidC), and the E. coli F-ATPase subunit b (Ecb). The engineered strains constructed in this paper provided more host choices for the production of recombinant proteins.
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影响因子：
4.6
作者：
Ting, Wan-Wen;Tan, Shih-, I;Ng, I-Son
通讯作者：
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影响因子：
6.4
作者：
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Mairhofer J
影响因子：
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作者：
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影响因子：
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作者：
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通讯作者：
Doyle DA
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作者：
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