Protein ligation

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.

Fig. 1.5 A Natural protein splicing, B Trans splicing with a split intein. The two fragments can be prepared separately and reassembled in vitro to form an active intein domain for protein ligation. C Central segment labeling using two different split inteins.

The principle of in vivo cyclization is based on the circular permutation of precursor proteins containing an intein (Fig. 1.6 C) [74, 75, 80, 81]. A naturally occurring split intein, DnaE from Synechocystis sp. PCC6803, was first successfully used for cyclization. However, similarly to the IPL/EPL approach, a mixture of linear and circular forms is obtained, presumably because of hydrolysis of an intermediate [73, 75]. On the other hand, artificially split inteins such as Pl-Pful, DnaB, and the RecA intein have been successfully applied for in vivo cyclization, and only circular forms were observed [80-82], suggesting that the circular permutation approach is more suitable for cyclization. Compared to the IPL/EPL or the TWIN system, in vivo cyclization does not require any external thiol group for cyclization, similarly to protein ligation with split inteins. Moreover, there are no undesired products, such as linear forms or polymers, originating from intermolecular reactions. 

Machova Z, Muhle C, Krauss U, Trehin R, Koch A, Merkle HP, Beck-Sickinger AG (2002) Cellular internalization of enhanced green fluorescent protein ligated to a human calcitonin-based carrier peptide. Chem Biochem 3 672-677. 

Three other components that my laboratory has identified and partially purified from Fraction 2 of reticulocytes, termed CF1-CF3, are involved in the degradation of proteins ligated to ubiquitin [24]. These are apparently subcomplexes of the 26S proteasome, a large ATP-dependent protease complex first described by Re-chsteiner and co-workers [25], CF3 is identical to the 20S proteasome core particle [26], while CFl and CF2 may be similar to the base and lid subcomplexes of the 19S regulatory particle of the 26S proteasome, described more recently by the Finley laboratory [27], In hindsight, the reason for finding subcomplexes, rather than the complete 26S complex in Fraction 2 was technical we have routinely prepared Fraction 2 from ATP-depleted reticulocytes [20], under which conditions the 26S proteasome dissociates to its subcomplexes. We found that incubation of the three subcomplexes in the presence of ATP promotes their assembly to the 26S proteasome [24, 26]. The role of ATP in the assembly of the 26S proteasome complex remains unknown. 

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.

In 2006 an alternative protein ligation was published employing the highly selective and fast Diels-Alder reaction to the synthesis of lipoproteins. This reaction has already been used for bioconjugation... 

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