排序方式: 共有56条查询结果,搜索用时 31 毫秒
1.
Manoj Cheriyan Chandra Sekhar Pedamallu Kazuo Tori Francine Perler 《The Journal of biological chemistry》2013,288(9):6202-6211
Inteins are naturally occurring intervening sequences that catalyze a protein splicing reaction resulting in intein excision and concatenation of the flanking polypeptides (exteins) with a native peptide bond. Inteins display a diversity of catalytic mechanisms within a highly conserved fold that is shared with hedgehog autoprocessing proteins. The unusual chemistry of inteins has afforded powerful biotechnology tools for controlling enzyme function upon splicing and allowing peptides of different origins to be coupled in a specific, time-defined manner. The extein sequences immediately flanking the intein affect splicing and can be defined as the intein substrate. Because of the enormous potential complexity of all possible flanking sequences, studying intein substrate specificity has been difficult. Therefore, we developed a genetic selection for splicing-dependent kanamycin resistance with no significant bias when six amino acids that immediately flanked the intein insertion site were randomized. We applied this selection to examine the sequence space of residues flanking the Nostoc punctiforme Npu DnaE intein and found that this intein efficiently splices a much wider range of sequences than previously thought, with little N-extein specificity and only two important C-extein positions. The novel selected extein sequences were sufficient to promote splicing in three unrelated proteins, confirming the generalizable nature of the specificity data and defining new potential insertion sites for any target. Kinetic analysis showed splicing rates with the selected exteins that were as fast or faster than the native extein, refuting past assumptions that the naturally selected flanking extein sequences are optimal for splicing. 相似文献
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The identification of inteins in viral genomes is becoming increasingly common. Inteins are selfish DNA elements found within coding regions of host proteins. Following translation, they catalyse their own excision and the formation of a peptide bond between the flanking protein regions. Many inteins also display homing endonuclease function. Here, the newly identified coccolithovirus intein is described and is predicted to have both self-splicing and homing endonuclease activity. The biochemical mechanism of its protein splicing activity is hypothesised, and the prevalence of the intein among natural coccolithovirus isolates is tested. 相似文献
4.
Unprecedented rates and efficiencies revealed for new natural split inteins from metagenomic sources
Carvajal-Vallejos P Pallissé R Mootz HD Schmidt SR 《The Journal of biological chemistry》2012,287(34):28686-28696
Inteins excise themselves out of precursor proteins by the protein splicing reaction and have emerged as valuable protein engineering tools in numerous and diverse biotechnological applications. Split inteins have recently attracted particular interest because of the opportunities associated with generating a protein from two separate polypeptides and with trans-cleavage applications made possible by split intein mutants. However, natural split inteins are rare and differ greatly in their usefulness with regard to the achievable rates and yields. Here we report the first functional characterization of new split inteins previously identified by bioinformatics from metagenomic sources. The N- and C-terminal fragments of the four inteins gp41-1, gp41-8, NrdJ-1, and IMPDH-1 were prepared as fusion constructs with model proteins. Upon incubation of complementary pairs, we observed trans-splicing reactions with unprecedented rates and yields for all four inteins. Furthermore, no side reactions were detectable, and the precursor constructs were consumed virtually quantitatively. The rate for the gp41-1 intein, the most active intein on all accounts, was k = 1.8 ± 0.5 × 10(-1) s(-1), which is ~10-fold faster than the rate reported for the Npu DnaE intein and gives rise to completed reactions within 20-30 s. No cross-reactivity in exogenous combinations was observed. Using C1A mutants, all inteins were efficient in the C-terminal cleavage reaction, albeit at lower rates. C-terminal cleavage could be performed under a wide range of reaction conditions and also in the absence of native extein residues flanking the intein. Thus, these inteins hold great potential for splicing and cleavage applications. 相似文献
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Zhenming Du Yangzhong Liu Yuchuan Zheng Scott McCallum John Dansereau Victoria Derbyshire Marlene Belfort Georges Belfort Patrick Van Roey Chunyu Wang 《Biomolecular NMR assignments》2008,2(2):111-113
The backbone and side chain resonance assignments of an engineered intein based on Mycobacterium tuberculosis RecA have been determined based on triple-resonance experiments with the uniformly [13C,15N]-labeled protein. 相似文献
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Expression of a cytotoxic cationic antibacterial peptide in Escherichia coli using two fusion partners 总被引:2,自引:0,他引:2
Chen YQ Zhang SQ Li BC Qiu W Jiao B Zhang J Diao ZY 《Protein expression and purification》2008,57(2):303-311
It has been reported that it is difficult to express cationic antibacterial peptides in engineered bacteria because such peptides are highly toxic to the host bacteria cells and sensitive to intracellular proteases. Antibacterial peptide CM4 (ABP-CM4) is a small cationic peptide with broad-spectrum activities against bacteria, fungi and tumor cells, which may possibly be used as an antimicrobial agent. Here we tried to express ABP-CM4 in Escherichia coli cells using either the GST fusion system or the intein-mediated fusion expression system. In order to investigate the possible use of these two fusion partners in cationic small peptide expression and purification, a mutant ABP-CMt, which is a highly positively charged peptide with +9 charges at neutral pH, was designed. In the present study, we have shown that both ABP-CM4 and ABP-CMt peptides can be expressed and purified by the intein-mediated expression system but not by the GST fusion expression system. Thus the intein-mediated peptide expression and purification system potentially could be employed for the production of recombinant protease-sensitive and cytotoxic peptides. 相似文献
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The DnaE intein of Synechocystis sp. PCC6803 (Ssp DnaE intein) is the first split intein identified in nature. Its N-terminal fragment (Int-n) is attached to the end of the N-terminal half of the DnaE protein (DnaE-n) to form the precursor DnaE-n/Int-n, while the C-terminal fragment (Int-c) precedes the C-terminal half of the DnaE protein (DnaE-c) to form the precursor Int-c/DnaE-c. Int-n and Int-c fragments in the separate precursors catalyze, in concert, a protein trans-splicing process to splice the flanking DnaE-n and DnaE-c into a functional catalytic subunit of DNA polymerase III. They then release themselves from the precursors. Previously, the Ssp DnaE intein has been used to reconstitute a protein trans-splicing mechanism in stably transformed Arabidopsis thaliana, resulting in successful reassembly of an intact and functional GUS from two halves of a split GUS protein. In this report, transient expression using a biolistic particle bombardment approach is described for functional analysis of Ssp DnaE intein. Analyses confirmed that the Ssp DnaE intein could catalyze protein trans-splicing not only in model plants but also in monocot and dicot crops. It also demonstrated that when up to 45 amino acid residues were removed from the C-terminus of the Int-n fragment, the Int-n fragment was still able to function in the protein trans-splicing process. 相似文献
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Attachment of an NMR-invisible solubility enhancement tag using a sortase-mediated protein ligation method 总被引:1,自引:1,他引:0
Yoshihiro Kobashigawa Hiroyuki Kumeta Kenji Ogura Fuyuhiko Inagaki 《Journal of biomolecular NMR》2009,43(3):145-150
Sample solubility is essential for structural studies of proteins by solution NMR. Attachment of a solubility enhancement
tag, such as GB1, MBP and thioredoxin, to a target protein has been used for this purpose. However, signal overlap of the
tag with the target protein often made the spectral analysis difficult. Here we report a sortase-mediated protein ligation
method to eliminate NMR signals arising from the tag by preparing the isotopically labeled target protein attached with the
non-labeled GB1 tag at the C-terminus.
Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users. 相似文献
9.
Ulrich Arnold 《Biotechnology letters》2009,31(8):1129-1139
The biotechnological application of enzymes necessitates a permanent quest for new biocatalysts. Among others, improvement
of catalytic activity, modification of substrate specificity, or increase in stability of the enzymes are desirable goals.
The exploration of homologous enzymes from various sources or DNA-based methods, like site-directed mutagenesis or directed
evolution, yield an incredible variety of biocatalysts but they all rely on the restricted number of canonical amino acids.
Chemistry offers an almost unlimited palette of additional modifications which can endow the proteins with improved or even
completely new properties. Numerous techniques to furnish proteins with non-natural amino acids or non-proteinogenic modules
have been introduced and are reviewed with special focus on expressed protein ligation, a method that combines the potential
of protein biosynthesis and chemical synthesis.
An erratum to this article can be found at 相似文献
10.
蛋白质内含子的研究进展 总被引:1,自引:0,他引:1
自1990年发现第一个蛋白质内含子以来,对其研究愈加引起注意。蛋白质内含子是蛋白质剪接元件,可从前体蛋白中切除并将两侧外显子连接起来成为成熟蛋白质,标准的蛋白质剪接主要包括四步亲核置换反应,新近又发现一种反式剪接机制。蛋白质内含子的演化形成存在先天遗传和后天插入两种方式,但目前还没有直接的实验证据。数据库显示,蛋白质内含子有10个保守模体:A、N2、B、N4、C、D、E、H、F和G,它们在蛋白质剪接过程中具有不同的作用。作为蛋白质剪切元件的蛋白质内含子,是蛋白质工程中一个功能强大的工具,具有重要的实践意义。现对蛋白质内含子的命名、分布、结构、剪接方式以及应用前景等作一全面的综述。 相似文献