Post-translational ligation

Staudinger ligation techniques also can be used to detect post-translational modification of proteins in vivo. Hang et al. (2007) developed a method to monitor fatty acid acylation of proteins using azido-fatty acids fed to cells. The two major types of fatty acid acylation,... 

Figure 17.20 An azido-palmitic acid derivative can be added to cells to obtain palmitoylated proteins that contain an azide group able to participate in the Staudinger ligation reaction. Biotinylation of these post-translationally modified sites then can be done in vivo using a biotin-phosphine reagent.

For the production purpose, abolishment of feedback control and fusion of sequential enzymes may be desirable. The recombinant technology is the method of choice when the redesigning involves substimtions between coded amino acids. However, for substitutions involving artificial, noncoded amino acids or their analogues/derivatives (e.g. post-translationally modified amino acids, isotopic label of specific residue), chemical methods in particular semi-chemical synthesis becomes necessary (Chaiken, 1981). The frag-ments/chains of modified, synthetic or expressed polypeptides are enzymatically or chemically ligated (Muir, 2003). 

Over the last 20 years, native chemical ligation has ushered in a new era in the total chemical synthesis of proteins by enabling the efficient, programmed constmction of native stmctures, including those bearing post-translational modifications, as... 

Ribosomal protein S6 from Escherichia coli undergoes a unique post-translation modification where up to six glutamic acid residues are ligated to the C-terminus. [82-84] RimK, also a member of carboxylate-amine/thiol Ugase superfamily, mediated this post-translahonal modification. [84] In vitro analysis of RimK s)rnthesis resulted in 46-mer (maximum length) of a-poly (L-Glu) at pH 9.0, 30 °C. The maximum chain length was pH dependent. Furthermore, RimK demonstrated strict substrate specificity for Glu. [72]... 

C-terminal peptide tiiioesters are used extensively in synthetic protein chemistry, especially for native chemical ligation (NCL) and other chemoselective reactions, which has inspired a search for robust synthetic strategies. Initially, peptide thioesters were mainly prepared using solid-phase peptide synthesis with amino acids N -protected with Boc (Boc-SPPS) [1-3], see Chapter 4. However, this technique requires specialized equipment for handling of hydrofluoric acid (HP) for release of the peptide from the resin, and it is therefore currently not used in many laboratories. Furthermore, the HP treatment is incompatible with many post-translational modiflcations such as glycosylations or phosphorylations [4]. Boc-SPPS is described thoroughly in Chapter 4. 

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