High throughput screening identifies disulfide isomerase DsbC as a very efficient partner for recombinant expression of small disulfide-rich proteins in E. coli.
High throughput screening identifies disulfide isomerase DsbC as a very efficient partner for recombinant expression of small disulfide-rich proteins in E. coli.
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DOI：
10.1186/1475-2859-12-37
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发表时间：
2013-04-22
影响因子：
6.4
通讯作者：
Dive V
中科院分区：
文献类型：
--
作者：
Nozach H;Fruchart-Gaillard C;Fenaille F;Beau F;Ramos OH;Douzi B;Saez NJ;Moutiez M;Servent D;Gondry M;Thaï R;Cuniasse P;Vincentelli R;Dive V
富含二硫化物的蛋白质或DRP是涵盖多种药理学、治疗学和/或生物技术应用的通用生物活性化合物。尽管如此，生产足够数量的DRP是其完整的结构或功能表征的主要瓶颈。在大肠杆菌中重组表达这种含有多个二硫键的小蛋白。大肠杆菌的方法被认为是困难的，并且一般的方法和方案，特别是在高通量规模上的方法和方案是有限的。在这里，我们报告了一种高通量筛选方法，该方法允许系统研究12种细胞质配偶体对菌株BL 21（DE 3）pLysS、Origami B（DE 3）pLysS和SHuffle® T7 Express lysY（1008条件）中28种DRP的溶解和折叠影响。筛选鉴定了导致选择用于研究的28种DRP的成功可溶性表达的条件。在每种细菌菌株测试的336种条件中，使用菌株BL 21（DE 3）pLysS在196种条件中检测到可溶性表达，而对于菌株Origami B（DE 3）pLysS和SHuffle® T7 Express lysY分别仅鉴定出44种和50种可溶性表达条件。为了评估DRP的氧化还原状态，将溶解度筛选与质谱法（MS）结合以确定所产生的DRP或融合蛋白的确切质量。为了验证在分析规模下获得的结果，沿着了几个以更大规模表达和纯化的蛋白质的实例及其MS和功能表征。我们的研究结果表明，可溶性和功能性DRPs的生产与细胞质的合作伙伴是可能的，在E。杆菌尽管其细胞质还原，但对于与增溶配偶体融合的DRP的产生，BL 21（DE 3）pLysS比Origami B（DE 3）pLysS和SHuffle® T7 Express lysY trx B-/戈尔-菌株更有效。然而，我们的数据表明，氧化的蛋白质发生离体。我们的协议允许使用DsbC作为融合伙伴生产大量多样性的DRP，在许多情况下导致毫克级的纯活性DRP。这些结果为研究和开发具有治疗或生物技术意义的DRPs开辟了新的可能性，这些DRPs的生产以前是一个限制。
Disulfide-rich proteins or DRPs are versatile bioactive compounds that encompass a wide variety of pharmacological, therapeutic, and/or biotechnological applications. Still, the production of DRPs in sufficient quantities is a major bottleneck for their complete structural or functional characterization. Recombinant expression of such small proteins containing multiple disulfide bonds in the bacteria E. coli is considered difficult and general methods and protocols, particularly on a high throughput scale, are limited. Here we report a high throughput screening approach that allowed the systematic investigation of the solubilizing and folding influence of twelve cytoplasmic partners on 28 DRPs in the strains BL21 (DE3) pLysS, Origami B (DE3) pLysS and SHuffle® T7 Express lysY (1008 conditions). The screening identified the conditions leading to the successful soluble expression of the 28 DRPs selected for the study. Amongst 336 conditions tested per bacterial strain, soluble expression was detected in 196 conditions using the strain BL21 (DE3) pLysS, whereas only 44 and 50 conditions for soluble expression were identified for the strains Origami B (DE3) pLysS and SHuffle® T7 Express lysY respectively. To assess the redox states of the DRPs, the solubility screen was coupled with mass spectrometry (MS) to determine the exact masses of the produced DRPs or fusion proteins. To validate the results obtained at analytical scale, several examples of proteins expressed and purified to a larger scale are presented along with their MS and functional characterization. Our results show that the production of soluble and functional DRPs with cytoplasmic partners is possible in E. coli. In spite of its reducing cytoplasm, BL21 (DE3) pLysS is more efficient than the Origami B (DE3) pLysS and SHuffle® T7 Express lysY trxB-/gor- strains for the production of DRPs in fusion with solubilizing partners. However, our data suggest that oxidation of the proteins occurs ex vivo. Our protocols allow the production of a large diversity of DRPs using DsbC as a fusion partner, leading to pure active DRPs at milligram scale in many cases. These results open up new possibilities for the study and development of DRPs with therapeutic or biotechnological interest whose production was previously a limitation.
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影响因子：
6.4
作者：
Hatahet F;Nguyen VD;Salo KE;Ruddock LW
通讯作者：
Ruddock LW
影响因子：
1.6
作者：
Drevet, P;Lemaire, C;Menez, A
通讯作者：
Menez, A
DOI：
10.1073/pnas.96.24.13703
发表时间：
1999-11-23
影响因子：
11.1
作者：
Bessette, PH;Åslund, F;Georgiou, G
通讯作者：
Georgiou, G
影响因子：
1.6
作者：
Bogomolovas, Julius;Simon, Bernd;Stier, Gunter
通讯作者：
Stier, Gunter
影响因子：
3.6
作者：
Fruchart-Gaillard, C;Mourier, G;Servent, D
通讯作者：
Servent, D