Recent advances in in vivo applications of intein-mediated protein splicing

Topilina, Natalya I.; Mills, Kenneth V.

doi:10.1186/1759-8753-5-5

Cited by 88 publications

(73 citation statements)

References 130 publications

(143 reference statements)

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“…CPS depends on the presence of a particular trigger, such as a change in redox state, temperature, small molecules, or light, as has been recently reviewed (7,8). The existence of stimulus-dependent inteins suggests the possibility that some inteins may adapt to their intracellular niche by becoming post-translational regulatory elements that modulate protein splicing in accord with environmental conditions.…”

Section: Conditional Protein Splicing and Splicing Regulationmentioning

confidence: 99%

Enigmatic Distribution, Evolution, and Function of Inteins

Новикова

Topilina

Belfort

2014

Journal of Biological Chemistry

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Inteins are mobile genetic elements capable of self-splicing post-translationally. They exist in all three domains of life including in viruses and bacteriophage, where they have a sporadic distribution even among very closely related species. In this review, we address this anomalous distribution from the point of view of the evolution of the host species as well as the intrinsic features of the inteins that contribute to their genetic mobility. We also discuss the incidence of inteins in functionally important sites of their host proteins. Finally, we describe instances of conditional protein splicing. These latter observations lead us to the hypothesis that some inteins have adapted to become sensors that play regulatory roles within their host protein, to the advantage of the organism in which they reside. Protein SplicingProtein splicing is a naturally occurring biochemical process that mediates the post-translational conversion of a precursor polypeptide into a mature and functional protein through the removal of an internal protein element, called an intein (Fig. 1A). The process is analogous to intron splicing at the RNA level. The protein splicing mechanism involves a series of autocatalytic peptide bond rearrangements, where the intein excises itself from the precursor polypeptide with concurrent ligation of the flanking sequences, called exteins (N-or C-exteins relative to the position of intein) (for review, see Refs. 1-3). As a result of this process, two proteins are produced from a single polypeptide product. The term intein refers to both the genetic element in the DNA or RNA and the protein splicing entity.Most inteins are expressed within a single polypeptide chain (cis-splicing inteins), but some are split into two polypeptides each containing one extein and an intein fragment (trans-splicing inteins) (4, 5). In the case of split inteins, reassociation of the fragments at a zipper-like interface (6) precedes protein splicing (Fig. 1B). Both cis-splicing and trans-splicing inteins are frequently utilized in various biotechnological applications including protein purification, modification, labeling, and posttranslational control of expressed proteins (reviewed in Refs. 7 and 8). The cis-splicing inteins often contain a distinct homing endonuclease (HEN) 2 domain (9). HEN-containing inteins are naturally occurring mobile genetic elements. The presence of a HEN provides inteins the ability to transfer their coding elements into homologous alleles at homing sites that lack the intein sequence (Fig. 1D). This HEN-mediated homing process can result in horizontal gene transfer (HGT) of inteins, by invasion of diverse species, followed by vertical transmission of inteins (10, 11). Moreover, HEN-containing inteins are involved in a so-called "homing cycle" that includes two opposing processes, precise intein loss and reinvasion of a newly formed vacant homing site. It is believed that the homing cycle allows the HEN to avoid fixation and functional decay in one locus (12).

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Section: Conditional Protein Splicing and Splicing Regulationmentioning

confidence: 99%

Enigmatic Distribution, Evolution, and Function of Inteins

Новикова

Topilina

Belfort

2014

Journal of Biological Chemistry

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“…Presumably, the larger VMA DNA sequence might result in a lower plasmid copy number, thereby resulting ultimately in lower levels of expression of both target proteins! It was previously viewed that intein precursors/ intermediates were only cleavable under optimized conditions furnished in vitro (Telenti et al 1997;Topilina and Mills 2014). Thus, disposable kits claimed to possess an optimal environment for the cleavage of intein precursors/ intermediates have been developed for commercial application (Wang et al 2010a).…”

Section: Discussionmentioning

confidence: 98%

“…Inteins, also known as protein introns, have been found to be present in many microbial species (Topilina and Mills 2014). They are products derived from self-cleavable precursors and/ or intermediates comprising inteins themselves and two adjacent components, designated N-exteins and C-exteins, which are located respectively at the N-and C-termini of inteins (Pietrokovski 1994;Wang et al 2010a;Elleuche and Pöggeler 2010;Li 2011;Volkmann and Mootz 2013).…”

Section: Introductionmentioning

confidence: 99%

Engineering versatile protein expression systems mediated by inteins in Escherichia coli

Kwong

Wong

2015

Appl Microbiol Biotechnol

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We have recently employed an intein, Saccharomyces cerevisiae vascular membrane ATPase (VMA), in conjunction with efficient expression and secretory functions formed between the ompA leader sequence and the human epidermal growth factor (EGF) gene (fused at the 5' end of VMA), and the human basic fibroblast growth factor (bFGF) gene (fused at the 3' end of VMA), to engineer an efficient intein-based Escherichia coli system for high-level co-expression of EGF and bFGF as authentic mature products. Both products were found not only excreted to the culture medium but also located, surprisingly, in the cytoplasm (Kwong and Wong 2013). In this study, we employed two structurally varied inteins, VMA and Mycobacterium xenopi GyraseA (GyrA), and further demonstrated that despite acting alone, both VMA and GyrA were able to mediate successful co-expression of two widely different proteins, EGF and an endoglucanase (Eng) in E. coli. Although EGF and Eng were initially expressed as large precursors/intermediates, they were soluble and auto-cleavable to finally yield the desired products in both the cytoplasm and culture media. The results further substantiate our postulation that the aforementioned intein/E. coli approach might lead to the development of cost-effective and versatile host systems, wherein all culture fractions are involved in producing the target proteins.

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“…We conclude that there is no competition between the two catalytic cysteines for attack on the N-terminal splice junction and that the Bvi IcmO intein is unable to splice by the class 2 mechanism. The Bvi IcmO intein expands the repertoire of potential insertion sites for class 3 inteins in target proteins to include Cys for the numerous in vivo and in vitro applications based on intein technology (Aranko et al 2014; Topilina and Mills 2014; Wood and Camarero 2014). Inteins continue to prove to be intriguing and robust single turnover enzymes.…”

Section: Discussionmentioning

confidence: 99%

Sequential formation of two branched intermediates during protein splicing of class three inteins

Tori

Perler

2016

Extremophiles

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Inteins are the protein equivalent of introns. They are seamlessly removed during post-translational maturation of their host protein (extein). Inteins from extremophiles played a key role in understanding intein-mediated protein splicing. There are currently three classes of inteins defined by catalytic mechanism and sequence signatures. This study demonstrates splicing of three class 3 mini-inteins: Burkholderia vietnamiensis G4 Bvi IcmO intein, Mycobacterium smegmatis MC2 155 Msm DnaB-1 intein and Mycobacterium leprae strain TN Mle DnaB intein. B. vietnamiensis has a broad ecological range and remediates trichloroethene. M. smegmatis is a biofilm forming soil bacteria. Although other intein classes have only a single branched intermediate at the C-terminal splice junction, the class 3 intein reaction pathway includes two branched intermediates. The class 3 specific branched intermediate is formed by an internal cysteine, while the C-terminal branch intermediate is at a serine or threonine in all class 3 inteins except the Bvi IcmO intein, where it is a cysteine. This latter cysteine was unable to compensate for mutation of the class 3-specific internal catalytic cysteine despite the Bvi IcmO intein having an N-terminal splice junction naturally tuned for a cysteine nucleophile, demonstrating the mandatory order of branch intermediates in class 3 inteins.

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Recent advances in in vivo applications of intein-mediated protein splicing

Cited by 88 publications

References 130 publications

Enigmatic Distribution, Evolution, and Function of Inteins

Enigmatic Distribution, Evolution, and Function of Inteins

Engineering versatile protein expression systems mediated by inteins in Escherichia coli

Sequential formation of two branched intermediates during protein splicing of class three inteins

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