Another method which uses a similar in vitro approach for cyclization of peptides and proteins is the two inteins system (TWIN) developed by New England Biolabs (Evans et al. 1999). TWIN uses two self-cleaving inteins, one with N-terminal cleavage activity (Mycobacterium xenopi GyrA intein), and the other with C-terminal cleavage activity (Ssp DnaB intein), to generate a protein possessing both an N-terminal cysteine and a C-terminal thioester. The fusion of CBDs to the inteins allows affinity purification of the precursor protein by chitin resin. The Ssp DnaB intein undergoes cleavage at its C-termi-nus by cyclization of Asnl54 (pH induced) whereas the Mxe GyrA intein undergoes thiol-induced (addition of MESNA) cleavage of the thioester bond (formed as a result of NIS acyl shift at cysteine). Upon release of the target protein the free thiol at the N-terminus reacts with the thioester of the C-ter-minus intramolecularly, leading to cyclization, or intermolecularly, resulting in polymerization (Fig. 2).

The cleavage efficiency was found to be somewhat determined by the amino acids adjacent to the scissile peptide (Evans et al. 1999). Significant in vivo cleavage of >50% was observed with the Cys-Gly-Ala sequence adjacent to the C-terminus of the Sspl DnaB mini-intein; this rate was reduced to <10% with Cys-Arg-Ala. Thus cleavage can be modulated through modification of the extein sequence without changing the intein.

The TWIN system has been used in the cyclization of several proteins and peptides (Evans et al. 1999). Thioredoxin (135 amino acids) was cyclized using a 12 amino acid (9 on the N-terminus, 3 on the C-terminus) linker and >80% of the isolated protein was found to be cyclic. Interestingly, increasing the length of the linker to 26 amino acids (3 on the N-teminus, 23 on the C-ter-minus) decreased the rate of cyclization to 50%. In another example, maltose-

binding protein (395 amino acids) also showed a 50% cyclization rate when a 26 amino acid linker was used.

Several cyclic peptides were also isolated including RGD (10 amino acids), an inhibitor of platelet aggregation (Yamamoto et al. 1995), and CDR-H3/C2 (16 amino acids; Levi et al. 1993), which inhibits HIV-1 replication. Previously, using chemical synthesis, these peptides were cyclized with cysteines at the N- and C-termini then oxidized to form a disulfide bond. The biological synthesis is postulated to be superior as the peptides are cyclized with a native peptide bond between the N- and C-termini, which is thought to endow resistance to reducing environments (such as the intracellular environment) and exoproteases.

Neither intein-mediated ligation nor TWIN allows for the total biasing of the final step toward cyclization or polymerization; although the rate of polymerization is concentration-dependent (increasing concentration increases the likelihood of an intermolecular reaction), other factors such as the protein structure, flexibility of the linkers and the termini, and the propensity to self-associate also determine the direction of the final step.

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