Expressed chemical ligation

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.

However, if the expressed protein is treated on the affinity support using thiophenol, this also will release the protein and result in a phenylthioester at its C-terminal, which is the reactive intermediate imminendy suitable for native chemical ligation. Treatment of this activated thioester protein with a N-terminal cysteine peptide induces the native chemical ligation reaction and couples the peptide to the expressed protein through an amide bond (Severinov and Muir, 1998) (Figure 17.27). 

EPL extends the applicability of native chemical ligation to recombinantly produced proteins using the mutant mini intein vector system. Proteins being expressed using this method will... 

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.

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.

The development of EPL has facilitated the production of large protein targets, but the requirement of specific N-terminal amino acids at the ligation site (cysteine [7], selenocysteine ]66J) reduces the general utilization of this method. Recently, we introduced a novel approach that we named expressed enzymatic ligation (EEL) for the semisynthesis of larger and chemically modified proteins that combines the advantages of the EPL with those of the substrate mimetic... 

Machova, Z., von Eggelkraut-Gottanka, R., Wehofsky, N., Bordusa, E., and Beck-Sickinger, A. G. (2003) Expressed enzymatic ligation a new approach for the synthesis of chemically modified proteins, Angew. Chem. Int. Ed. 42, 4916-4918. 

The technique of native chemical ligation (NCL) or expressed protein ligation (EPL) (42) tremendously expanded the scope of peptide/protein synthesis. This approach also has been adopted for the construction of neoglycoproteins. For complete synthetic... 

Dawson PE, Muir TW, Clarklewis I, Kent SBH. Synthesis of proteins by native chemical ligation. Science 1994 266 776-779. Hofmann RM, Muir TW Recent advances in the application of expressed protein ligation to protein engineering. Curr. Opin. Biotechnol. 2002 13 297-303. 

Keywords Biophysical monitors Chemical synthesis of proteins Copper binding Expressed protein ligation Interaction of PrP with small molecules... 

EPL Expressed protein ligation NCL Native chemical ligation PrP Prion protein... 

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