Inteins, expressed protein

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.

Fusion protein containing the expressed protein, the mutant intein, and the CBD... 

Intein cleavage and release of expressed protein with N-terminal Cys... 

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.

Fig. 1.4 A I ntein-mediated protein ligation (IPL) (also called expressed protein ligation EPL). B The chemical mechanism of protein cyclization by the IPL/EPL approach. HAC denotes the sequence of the active site of the intein, e.g. His-Ala-Cys. CBD stands for chitin-binding domain.

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... 

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). 

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...

Evans, T. C. Jr., Benner, J., and Xu, M. Q. (1999) The cyclization and polymerization of bacteiially expressed proteins using modified self-splicing inteins. J. Biol. Chem. 274, 18,359-18,363. 

Harnessing protein splicing, researchers now have the ability to generate recombinant protein a-thioesters through the thiolysis of an appropriately mutated protein-intein fusion. In principle, this means that synthetic and recombinant building blocks can be fused in a semisynthetic version of NCL. Such an approach was first reported in 1998 and has been named expressed protein ligation [8],... 

Fig. 10.1-3 Expressed protein ligation. as a fusion to the N-terminus of an intein. Synthesis of recombinant protein thioesters The CBD allows for purification. The usingthe IMPACT system. Thioesters are thioester resulting from thiolysis can be obtained by expressing a protein of interest ligated under the conditions of NCL...

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