Native proteins by chemical ligation

Dawson, P.E., and Kent, S.B. (2000) Synthesis of native proteins by chemical ligation. Annu. Rev. Biochem. 69, 923-960. 

Dawson PE, Kent SB. Synthesis of native proteins by chemical ligation. 

Fig. 14 Molecular structure of synthetic erythropoietin protein (SEP). A Primary amino acid sequence with three ligation sites circled in red. B Structure of the branched, negatively charged poly(ethylene glycol)-based polymer. C Scheme for the synthesis of SEP by chemical ligation. Branched polymers were first attached to the individual peptide segments by oxime-forming ligation followed by native chemical ligation. Reprinted with permission from [62]. Copyright 2003 AAAS...

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+.

The following protocol for EPL, including purification using a CBD fusion tag followed by native chemical ligation, is based on the methods of Muir et al. (1998), Chong et al. (1997, 1998), Evans et al. (1998), Severinov and Muir (1998), and the NEB instruction manual for the IMPACT-TWIN system. The recombinant protein is recovered from the affinity column as the thioester derivative ready for reaction with a N-terminal Cys peptide or another tag containing a Cys residue. 

Beligere, G.S., and Dawson, P.E. (1999) Synthesis of a three zinc finger protein, Zif268, by native chemical ligation. Biopolymers 51(5), 363-369. 

Pal, G., Santamaria, F., Kossiakoff, A.A., and Lu, W. (2003) The first semi-synthetic serine protease made by native chemical ligation. Protein Expr. Purif. 29(2), 185-192. 

P.E. Dawson, T.W. Muir, I. Clark-Lewis, S.B.H. Kent, Synthesis of proteins by native chemical ligation. Science 266(5186) (1994) 776-779. 

Hackeng, T. M., Griffin, J. H., and Dawson, P. E. (1999) Protein synthesis by native chemical ligation expanded scope by using straightforward methodology. Proc. Natl. Acad. Sci. USA 96, 10,068-10,073. 

Yan, L. Z. and Dawson, P. E. (2001) Synthesis of peptides and proteins without cysteine residues by native chemical ligation combined with desulfurization. J. Am. Chem. Soc. 123, 526-533. 

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