Intein tags

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+.

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.

The modified Mth RIRl, Mxe GyrA, and Ssp DnaB mini-inteins have been recently applied to the isolation of proteins with an N-terminal cysteine residues (29,30). These inteins undergo temperature- and pH-dependent C-termi-nal cleavage when the N-terminal cysteine residue of the intein is substituted with alanine (Table 2). The target protein is recombinantly expressed as a fusion protein with the C-terminal intein tag (31) (Fig. 3B). After intein splicing the protein that possesses N-terminal cysteine is generated. Moreover, such a protein can be obtained by total chemical synthesis and different chemical labels or non-canonical amino acids can be site-specifically incorporated into the sequence. 

Gao et took this technique further by polymerizing from the G-terminus of GFP via ATRP. GFP was expressed to contain a Mycobacterium xenopi GyrA (Mxe)-intein tag... 

This ubiquitin intein system can also be utilized to make a DUB substrate rather than inhibitors by attaching a C-terminal fluorescent tag such as 7-amidomethylcoumarin (AMC) instead of vinyl sulfone. DUBs cleave the ubiquitin derivative and release the fluorescent tag, a process that can be followed fluoromet-rically. Eluorometric assays can then be used to determine a particular DUB s preferred substrate or to quantitate DUB activity in crude lysates. AMC substrates... 

His-tagged GUS-fusion proteins have been produced and isolated from tobacco chloroplasts. His-tagged proteins have also been extracted by foam frachonahon (Crofcheck et al., 2003,2004) or by a modihed intein expression system (Morassutti et al., 2002). 

Proteins containing selfcleaving/controlled phase separation tags (inteins)... 

Key Words Expressed protein ligation (EPL) intein protein engineering protein splicing purification tag. 

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. 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.

In addition to the commonly used TEV and 3 c proteases, several other proteases are commonly used to remove fusion tags (see Table 2). There are also other tag removal systems that do not rely on proteases. For example, the intein system is an autocatalytic cleavage of the produced fusion protein.297 For SUMO fusion proteins there are several hydrolases available for tag removal. Because the SUMO hydrolases recognize the whole fold of the tag and not just an amino acid sequence, they are highly specific and not prone to unintended cleavage.94 296... 

The real beauty of this system is that the self-cleavage reaction can be performed on an affinity column directly after purification on cellulose beads, so that the protein of interest can be eluted directly without any attached protein affinity tags. Naturally, fusion proteins may be engineered with an intein protein affinity tag fused at either the N- or C-terminus of the protein of interest, as appropriate. 

Figure 8.8 Expressed protein ligation. The final step of protein splicing by the intein is inactivated by the mutation of the C-tenninal Asn to Ala. Proteins expressed as in-frame N-tenninal fusions to such mutant inteins can be cleaved by thiols to give corresponding protein (N-peptide) thioester derivatives. The tagged inteins are removed. The N-peptide thioesters can then react with an aCys-containing peptide (C-peptide)...

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