Thioester intein

Figure 17.13 Expressed proteins containing a thioester intein tag can be specifically modified using a cysteine-alkyne derivative by transthioesterification followed by an internal S - N shift.

Figure 17.14 An expressed protein containing a thioester intein tag that was subsequently modified by native chemical ligation to contain an alkyne group then can be labeled using an azido-fluorescein probe by the click chemistry reaction in the presence of Cu1+.

Muir et al. (1998) realized that the intein reaction could be used to facilitate a native chemical ligation with a synthetic N-terminal cysteine-containing peptide or cysteine-containing molecule. With the discovery of a mutant intein that could form an intermediate thioester but not go on to complete the splice and ligation reaction (Xu and Perler, 1996 Chong et al.,... 

Figure 17.27 The EPL process involves a fusion protein containing an intein tag plus a CBD. The fusion protein is captured on an immobilized chitin resin and after removal of contaminating proteins, it is eluted using thiophenol, which cleaves at the thioester bond between the intein and the desired expressed protein. This releases a phenylth-ioester-activated protein that can be used in the native chemical ligation reaction with another peptide containing an N-terminal cysteine residue. Conjugation results in a native amide (peptide) bond formed between them.

Figure 17.28 EPL reactions can be used to couple a fusion protein to a surface containing a thioester derivative. After cells are grown and the fusion protein expressed, a pH and temperature shift causes intein cleavage with release of the expressed protein with an N-terminal cysteine residue. Reaction with the thioester surface results in a native chemical ligation reaction that forms an amide bond linkage with the expressed protein.

Figure 17.29 An expressed protein containing a mutant intein segment can undergo self cleavage to form an N-terminal cysteine residue, which then can be reacted with a thioester probe to label specifically the protein via an amide bond.

A full-length human csk DNA that codes for Csk, a 50-kDa protein that catalyzes the phosphorylation of a tyrosine within the C-terminal tail of Srk, was inserted in a plasmid and in-frame with a modified intein-chitin binding domain (CBD) encoding sequence, where intein is a protein-splicing element. 130 The expressed protein underwent a conversion into a thioester of the N-terminal cysteine of the intein, but the normal second step of an intein splicing did not occur. The protein was then bound to a chitin resin and washed. The resin containing the bound protein was treated with 2% benzenethiol to cleave the thioester and give the free phenyl thioester, which was immediately treated with one of the synthetic... 

Native chemical ligation, which is described in Section 5.3, has proven to be a generally useful route to the total synthesis of proteins of up to medium size. A recent adaptation which utilizes a protein splicing element, an intein, to generate a reactive thioester at the C-terminus of a recombinant protein provides an interesting variant of this technique. 4,5 ... 

The splicing mechanism, which is illustrated for this intein, is shown in the accompanying equations.1 1 Step a is an N —> S or N —> O acyl shift. This is followed by transesterification (step b) which involves either thioesters (as illustrated) or oxygen esters. Formation of a succinmide intermediate (step c) releases the intein and the spliced protein. The latter must undergo an S—> N or O—> N acyl shift (step d), and the succinimide in the extein must be hydrolyzed to complete the process. 

Fig. 2. Scheme of protein splieing. Cleavage pathway proposed for intein that possesses a cysteine residue in eaeh spliee junetion. In the initial step a linear thioester intermediate is formed by an N-S acyl rearrangement at Cysi (N-terminal amino acid of the intein). Next, traw -thioesterification that involves nucleophilic attack of the side-... 

The efficiency of the intein cleavage varies with the type of intein used for fusion and with the cleavage conditions (pH, temperature). Since usage of different thiol compounds results not only in a diverse splicing activity but also in a diverse rate of thioester hydrolysis, both parameters have to be taken into account. Specific protein properties, like the structure or the sequence at the C-terminus of the target protein, may also affect the cleavage efficiency (28,29). 

Fig. 2. Continued) chain of Cys+i (N-terminal amino acid of the C-extein) on the thioester results in the formation of a branched intermediate. Excision of the intein occurs by peptide bond cleavage coupled to succinimide formation at the C-terminal asparagine of the intein. The ligated exteins undergo a spontaneous S-N acyl rearrangement to create a stable amide bond.

Peptide bond cleavage at either the N- or C-terminal splice junction can occur independently. Replacement of Cysj to Alai allows only the C-terminal intein splicing and leads to C-extein that includes the cysteine at its N-terminus. Alternatively, the cleavage at N-terminus of the intein takes place by the nucleophilic attack at the thioester intermediate formed when the C-terminal Asn of the intein is substituted by Ala. 

Fig. 3. Intein-mediated protein ligation. The IMPACT system allows affinity purification of proteins fused to an intein-CBD tag and their further isolation with a C-terminal thioester moiety (A), or an N-terminal cysteine (B). (A), N-terminal intein splicing for thioester isolation. Target protein (protein 1) is expressed in E. coli with C-terminally located intein-CBD tag. After specific binding to the chitin resin, the thiol reagent provokes the cleavage of the peptide bond between the target protein and the intein. Whereas the intein-CBD tag remains bound to the chitin resin, the protein thioester is eluted from the column. (B), C-terminal cleavage to obtain N-terminally...

Fig. 3. (Continued) cysteine-possessing protein. The protein of interest (protein 2) is expressed as CBD-intein-protein 2 precursor and purified by the chitin beads. Temperature- or pH-induced intein cleavage results in proteins with an N-terminal cysteine residue. Finally, the EPL (dotted line) of the protein thioester and the cysteine containing protein, which can be also obtained synthetically, proceeds under NCL conditions.

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.

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