Enzymatic ligation for synthesis of single-chain analogue of monellin by transglutaminase.

Monellin, a sweet protein, consists of two noncovalently associated polypeptide chains: an A chain of 44 amino acid residues and a B chain of 50 residues. Microbial transglutaminase (MTGase) was used for ligation of the monellin subunits without any protecting groups, and without activation of the C alpha-carboxyl group at the C-terminus. Since a peptide fragment LLQG is a good substrate for MTGase to form an amide bond between the gamma-amide group of the Gln residue and the epsilon-amino group of Lys, a monellin B chain analogue in which LLQG was elongated at the C-terminus (B-LLQG) was synthesized by solid-phase synthesis. The monellin A chain analogue in which KGK was elongated at the N-terminus (KGK-A) was synthesized by the same method as that of the B chain analogue. The KGK-A chain and the B-LLQG chain were coupled by MTGase to give single-chain analogue of monellin. The single-chain analogue of monellin was characterized by analytical reverse phase high performance liquid chromatography, electrospray ionization, and amino acid analyses. All analyses gave satisfactory results. The single-chain analogue of monellin was more heat stable than natural monellin.