Expression protein ligation, expressed

Muii T.W. (2003) Semisynthesis of proteins by expressed protein ligation. Ann. Rev. Biochem. 72, 249-289. 

Muir, T.W., Sondhi, D., and Cole, P.A. (1998) Expressed protein ligation A general method for protein engineering. Proc. Natl. Acad. Sci. USA 95, 6705-6710. 

Severinov, K., and Muir, T.W. (1998) Expressed protein ligation, a novel method for studying protein-protein interactions in transcription./. Biol. Chem. 273(26), 16205-16209. 

Inteln-Mediated Protein Ligation (IPL)/Expressed Protein Ligation (EPL) using the IMPACT System... 

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.

Scheme 33 Expressed protein ligation (EPL) as an example for a protein ligation method generating a native bond (Left). MIC ligation generating a non-native bond between the two coupied segments (right).

For Ras proteins, there is no chaperone protein available or known to solubilize the lipidated protein thus the refolding of, for example, lipidated N- and H-Ras is not feasible. However, lipidated K-Ras 110 can be generated via expressed protein ligation (EPL), since the polybasic C-terminal region of this protein helps to solubilize the protein even when farnesylated and facilitates the purification (Scheme 36). ... 

Scheme 36 Expressed protein ligation of the farnesylated K-Ras 4B C-terminus.

Blaschke, U. K., Silberstein, J., and Muir, T. W. (2000) Protein engineering by expressed protein ligation. Methods Enzymol. 328, 478-496. 

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...

Protein Engineering by Expressed Protein Ligation 3.1. Site-Specific Protein Modifications... 

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