Split inteins

Fig. 1.5 A Natural protein splicing, B Trans splicing with a split intein. The two fragments can be prepared separately and reassembled in vitro to form an active intein domain for protein ligation. C Central segment labeling using two different split inteins.

C) central-segment labeling with two split inteins... 

The principle of in vivo cyclization is based on the circular permutation of precursor proteins containing an intein (Fig. 1.6 C) [74, 75, 80, 81]. A naturally occurring split intein, DnaE from Synechocystis sp. PCC6803, was first successfully used for cyclization. However, similarly to the IPL/EPL approach, a mixture of linear and circular forms is obtained, presumably because of hydrolysis of an intermediate [73, 75]. On the other hand, artificially split inteins such as Pl-Pful, DnaB, and the RecA intein have been successfully applied for in vivo cyclization, and only circular forms were observed [80-82], suggesting that the circular permutation approach is more suitable for cyclization. Compared to the IPL/EPL or the TWIN system, in vivo cyclization does not require any external thiol group for cyclization, similarly to protein ligation with split inteins. Moreover, there are no undesired products, such as linear forms or polymers, originating from intermolecular reactions. 

Fig. 6. Cyclization and polymerization of proteins. Two approaches that employ inteins for the generation of circular recombinant protein, split intein system (A), and TWIN system (B), are demonstrated. (A), The target protein is inserted between the C-terminal intein (C-intein) and the N-terminal intein (N-intein) segment. After spontaneous intein assembly, the standard splicing reaction results in excised intein and cyclized target protein. (B), The two intein systems sandwich the target protein between two intein-CBD tags. Controlled C- and N-terminal intein cleavages lead to target protein owning both N-terminal cysteine and C-terminal thioester. Whereas the intramolecular condensation forms cycUzed proteins, intermolecular reaction gives dimeric and polymeric proteins.

Fig. 8. Protein-protein interaction study based on split intein. In order to monitor the protein interaction in vivo, the N- and C-terminal halves of the intein (N-intein and C-intein) are fused to N- and C-terminal halves of EGFP (A), or luciferase (B). Each of these fusion proteins is linked to the protein of interest (protein A) and its target protein (protein B). Upon protein A-protein B cooperation, the closely oriented intein fragments mediate intein splicing. The measurement of fluorescence intensity originated from the reconstituted mature EGFP protein or measurement of luciferase luminescence is possible.

Evans, T. C. Jr., Martin, D., Kolly, R., et al. (2000) Protein trans-splicing and cyclization by a naturally split intein from the dnaE gene of Synechocystis species PCC6803. /. Biol. Chem. 275, 9091-9094. 

Split-Inteins for Protein Semisynthesis in vitro and in vivo... 

The DnaE intein from Synechocystis ssp. is a naturally occurring split intein and consists of a longer N-terminal segment (123 amino acids) that can be C-terminally fused to almost any given protein sequence and expressed. The C-terminal segment... 

To extend the utility of the DnaB split intein, Liu et al. have tested 13 different sites to split this intein into two segments of different length (58). Until this series of experiments, all known artificial split inteins had been split at the endonuclease domain. Out of 13 tested sites, 3 gave functional split inteins that would undergo frawi -splicing, including 1 that consisted of only llN-terminal amino acids. Such a short N-terminal split intein half is accessible by chemical synthesis, and the introduction of chemically modified peptides at the N-terminus via frawi -splicing was recently demonstrated. Such a system nicely complements the already established C-terminal modification approach via the DnaE split intein (74). 

Wu H, Hu ZM, Liu XQ. Protein trans-splicing by a split intein encoded in a spht dnae gene of synechocystis sp. Pcc6803. Proc. Natl. Acad. Sci. U.S.A. 1998 95(16) 9226-9231. 

Shi J, Muir TW. Development of a tandem protein trans-splicing system based on native and engineered split inteins. J. Am. Chem. Soc. 2005 127(17) 6198-6206. 

Brenzel S, Kurpiers T, Mootz HD. Engineering artificially split inteins for appUcations in protein chemistry biochemical characterization of the spUt Ssp DnaB intein and comparison to the split See VMA intein. Biochemistry 2006 45(6) 1571—1578. 

The discovery of split inteins further advanced the scope of EPL into the realm of conditional protein splicing. As outlined above, certain inteins can be split into two halves. An advantage for a general applicability concerns the observation that the split site is asymmetric. This opens an access to chemical synthesis of the smaller intein half and hence an easy route to C-terminal modification of proteins with, e.g., fluorophores, lipid anchor, or other posttranslational modifications of proteins. 

N-terminal protein ligation, an approach towards native protein ligation using split inteins. The protein of interest is obtained by ligation of a recombinant and a synthetic fragment. The C-terminal part is obtained by recombinant synthesis from a fusion protein containing the C-extein (C-terminal part of the target protein) and the... 

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