Expressed enzymatic ligation

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. 

Over the last four decades, synthetic non-enzymatic oligonucleotide ligations have been explored as potential models for the evolution of replicating systems on the early Earth [2-11]. These studies have explored ligation reactions that occur on a template without protein/enzymatic catalysis. Such systems have both provided mechanisms for the early evolution of biopolymer catalysts and placed limits on template directed ligation. More recently, the lure of antisense molecules capable of disrupting specific gene expression has resulted in the exploration of backbone-modified nucleic acids [12-33]. 

In this view, several different strategies have been pursued to incorporate noncoded amino acids, including peptide synthesis [7] native chemical ligation [8, 9], enzyme-catalyzed semisynthesis [10, 11], biosynthetic incorporation via auxotrophic bacterial strain expression [12,13], and nonsense suppression methodologies in cell-free [4] or whole-cell [14] expression systems. Both native chemical ligation and enzymatic semi-synthesis require careful tailoring of the experimental procedures, whereas biosynthetic incorporation methods are not site-specific and are... 

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). 

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