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Optimizing Periplasmic Expression in Escherichia coli for the Production of Recombinant Proteins Tagged with the Small Metal-Binding Protein SmbP

机译:优化大肠杆菌中的周质表达,用于用小金属结合蛋白SMBP制备重组蛋白的生产

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We have previously shown that the small metal-binding protein (SmbP) extracted from the gram-negative bacterium Nitrosomonas europaeacan be employed as a fusion protein for the expression and purification of recombinant proteins inEscherichia coli.With the goal of increasing the amounts of SmbP-tagged proteins produced in theE. coli periplasm, we replaced the native SmbP signal peptide with three different signal sequences: two were from the proteins CusF and PelB, for transport via the Sec pathway, and one was the signal peptide from TorA, for transport via the Tat pathway. Expression of SmbP-tagged Red Fluorescent Protein (RFP) using these three alternative signal peptides individually showed a considerable increase in protein levels in the periplasm ofE. colias compared to its level using the SmbP signal sequence. Therefore, for routine periplasmic expression and purification of recombinant proteins inE. coli,we highly recommend the use of the fusion proteins PelB-SmbP or CusF-SmbP, since these signal sequences increase periplasmic production considerably as compared to the wild-type. Our work, finally, demonstrates that periplasmic expression for SmbP-tagged proteins is not limited to the Sec pathway, in that the TorA-SmbP construct can export reasonable quantities of folded proteins to the periplasm. Although the Sec route has been the most widely used, sometimes, depending on the nature of the protein of interest, for example, if it contains cofactors, it is more appropriate to consider using the Tat route over the Sec. SmbP therefore can be recommended in terms of its particular versatility when combined with signal peptides for the two different routes.
机译:我们先前已经表明,从革兰氏阴性菌硝基核糖胺蛋白酶中提取的小金属结合蛋白(SMBP)作为融合蛋白,用于表达和纯化重组蛋白质中的Coli。增加了SMBP的量标记的蛋白质在你中产生。 Coli Periplasm,我们用三种不同的信号序列取代了本地SMBP信号肽:两种来自蛋白质CUSF和PELB,通过SEC途径运输,并且一种是来自托拉的信号肽,通过TAT途径运输。使用这三种替代信号肽的SMBP标记的红色荧光蛋白(RFP)的表达单独地显示出蛋白质水平的显着增加。 COLIAS使用SMBP信号序列与其电平相比。因此,对于常规的周质表达和重组蛋白ine的纯化。大肠杆菌,我们强烈建议使用融合蛋白PELB-SMBP或CUSF-SMBP,因为与野生型相比,这些信号序列会显着增加周质产生。最后,我们的工作证明了SMBP标记蛋白的周质表达不限于SEC途径,因为Tora-SMBP构建体可以将合理的折叠蛋白质出口到周质上。虽然SEC途径是最广泛使用的,有时候,根据感兴趣的蛋白质的性质,例如,如果它包含辅助弧剂,则更适合考虑使用TAT路线在SEC上。因此,可以在与两种不同路线的信号肽结合时,在其特定的多功能性方面建议使用SMBP。

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