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^α‐carboxyl group at the C‐terminus. Since a peptide fragment LLQG is a good substrate for MTGase to form an amide bond between the γ‐amide group of the Gln residue and the ε‐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. © 1999 John Wiley & Sons, Inc. Biopoly 50: 193–200, 1999