Intein Mediated Ligation

A technique known as intein-mediated ligation (Iwai and Pluckthun 1999) allows for the in vitro cyclization of proteins. In this method the protein to be cyclized is fused at its C-terminus to a disabled Saccharomyces cerevisiae VMA intein (Hirata et al. 1990; Kane et al. 1990), which is itself fused at its C-terminus to a chitin-binding domain (CBD; Chong et al. 1997). The intein is disabled by mutation of the C-terminal asparagine residue to alanine, thus disabling the splicing function at the C-terminus. The N-terminus of the protein of interest is altered if necessary, so that the second residue of the protein is cysteine. This relies on the in vivo activity of E. coli methionyl-ami-nopeptidase (Hirel et al. 1989) to remove the terminal methionine resulting in an N-terminal cysteine. After expression the fusion protein is then captured on chitin affinity resin. The partially functioning intein catalyzes an N/S acyl shift, which replaces the peptide bond between the protein and the intein with a thioester bond. Addition of an external nucleophile such as MESNA (2-mer-captoethanesulfonic acid) attacks the thioester, resulting in cleavage of the fusion protein (Xu and Evans 2001).

A linear protein contains an N-terminal cysteine and a C-terminal thioester, which can react to form a peptide bond in either an inter- or intramolecular fashion yielding a polymeric or a cyclized protein, respectively. A drawback of this methodology is that the intramolecular cyclization has to compete with intermolecular processes such as polymerization and hydrolysis of the thioester group thus reducing the yield and complicating purification of the cyclic protein (Fig. 2).

An example of the application of this technique is the cyclization of TEM-1 lactamase (BLA; Iwai and Pluckthun 1999). The proximity of the N- and C-

Fig. 2. Intein-mediated ligation and TWIN. Intein-mediated ligation relies on the removal of the initial methionine by MAP which results in an N-terminal cysteine. In the TWIN system the N-terminal nucleophile is unmasked by the processing of the C-termi-nal portion of intein 1. In both cases the N-terminal intein undergoes an N/S acyl shift followed by thiol-induced cleavage by an external nucleophile. The N-terminal cysteine can react intramolecularly with the C-terminal thioester; the product undergoes a further S/N acyl shift forming the cyclic protein

Fig. 2. Intein-mediated ligation and TWIN. Intein-mediated ligation relies on the removal of the initial methionine by MAP which results in an N-terminal cysteine. In the TWIN system the N-terminal nucleophile is unmasked by the processing of the C-termi-nal portion of intein 1. In both cases the N-terminal intein undergoes an N/S acyl shift followed by thiol-induced cleavage by an external nucleophile. The N-terminal cysteine can react intramolecularly with the C-terminal thioester; the product undergoes a further S/N acyl shift forming the cyclic protein termini of BLA and the addition of a 15 amino acid linker allow its cycliza-tion. The circular form of BLA was found to be more stable to irreversible aggregation upon heating than the linear form. The linear protein had fully precipitated after heating at 50 °C for 1 h, whereas 50% of the cyclic form stayed in the soluble fraction; the midpoint of the transition (Tm) increased by 5 °C upon cyclization (45 °C for linear, 50 °C for cyclic). This difference was utilized in the purification of the cyclic form away from linear and polymerized forms of BLA. As the free energies of related proteins are often well correlated with the aggregation temperature, the stabilizing effect of cyclization is consistent with the decreased conformational entropy of the unfolded state, as predicted by polymer theory.

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