A dual-intein autoprocessing domain that directs synchronized protein co-expression in both prokaryotes and eukaryotes.
A dual-intein autoprocessing domain that directs synchronized protein co-expression in both prokaryotes and eukaryotes.
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DOI：
10.1038/srep08541
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发表时间：
2015-02-25
影响因子：
4.6
通讯作者：
Su WW
中科院分区：
文献类型：
--
作者：
Zhang B;Rapolu M;Liang Z;Han Z;Williams PG;Su WW
能够协调多个蛋白质的共表达对于各种重要的应用是必要的，例如蛋白质复合物的组装、性状堆叠和代谢工程。目前，多种重组蛋白共表达的方法很少，而且大多数方法不适用于原核和真核宿主。在这里，我们报告了一种新的多蛋白载体系统，该系统是基于一对自我切除的迷你内含肽串联融合，称为双内含肽（DI）结构域，以实现多个蛋白质的同步共表达。DI结构域包含通过肽接头串联连接的Ssp DnaE小型内含肽N159A突变体和Ssp DnaB小型内含肽C1A突变体，以通过自催化切割介导侧翼蛋白的有效释放。基本上完全释放的组成蛋白，GFP和RFP（mCherry），从一个多蛋白前体，在细菌，哺乳动物和植物宿主被证明。此外，GFP与氯霉素乙酰转移酶和硫氧还蛋白与RFP分别成功共表达，进一步证实了DI多蛋白系统的普遍适用性。总的来说，我们的研究结果表明，基于DI的多蛋白质技术是对多蛋白质共表达分子工具箱的一个非常有价值的补充，在生物技术、生物科学和生物医学中有着广泛的应用。
Being able to coordinate co-expression of multiple proteins is necessary for a variety of important applications such as assembly of protein complexes, trait stacking, and metabolic engineering. Currently only few options are available for multiple recombinant protein co-expression, and most of them are not applicable to both prokaryotic and eukaryotic hosts. Here, we report a new polyprotein vector system that is based on a pair of self-excising mini-inteins fused in tandem, termed the dual-intein (DI) domain, to achieve synchronized co-expression of multiple proteins. The DI domain comprises an Ssp DnaE mini-intein N159A mutant and an Ssp DnaB mini-intein C1A mutant connected in tandem by a peptide linker to mediate efficient release of the flanking proteins via autocatalytic cleavage. Essentially complete release of constituent proteins, GFP and RFP (mCherry), from a polyprotein precursor, in bacterial, mammalian, and plant hosts was demonstrated. In addition, successful co-expression of GFP with chloramphenicol acetyltransferase, and thioredoxin with RFP, respectively, further substantiates the general applicability of the DI polyprotein system. Collectively, our results demonstrate the DI-based polyprotein technology as a highly valuable addition to the molecular toolbox for multi-protein co-expression which finds vast applications in biotechnology, biosciences, and biomedicine.
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影响因子：
13.8
作者：
Bieniossek C;Imasaki T;Takagi Y;Berger I
通讯作者：
Berger I
影响因子：
6.4
作者：
Martínez-Alonso M;García-Fruitós E;Ferrer-Miralles N;Rinas U;Villaverde A
通讯作者：
Villaverde A
影响因子：
8.4
作者：
Brazier-Hicks, Melissa;Edwards, Robert
通讯作者：
Edwards, Robert
影响因子：
7.2
作者：
NI, M;CUI, D;GELVIN, SB
通讯作者：
GELVIN, SB
DOI：
10.1073/pnas.0904366106
发表时间：
2009-07-07
影响因子：
11.1
作者：
Amitai, Gil;Callahan, Brian P.;Belfort, Marlene
通讯作者：
Belfort, Marlene