Tandem peptide ligation for synthetic and natural biologicals.
We describe the concept and methods of peptide ligation and tandem peptide ligation for preparing synthetic and natural biologicals. Peptide ligation is a segment coupling method for free peptides or proteins through an amide bond without the use of a coupling reagent or a protecting group scheme. Because unprotected peptides or proteins prepared from either a chemical or biochemical source are being used as building blocks, the ligation removes the size limitation for peptide and protein synthesis. A key feature of the peptide ligation is that the coupling reaction is orthogonal, i.e. it is specific to a particular alpha-amino terminus (NT). This NT-amino acid-specific feature permits the development of a tandem peptide ligation method employing three unprotected peptide segments containing different NT-amino acids to form consecutively two amide bonds, an Xaa-SPro (thiaproline) and then an Xaa-Cys. This strategy was tested in peptides ranging from 28 to 70 amino acid residues, including analogues of somatostatins and two CC-chemokines MIP-1alpha and MIP-1beta. The thiaproline replacements in these peptides and proteins did not result in altered biological activity. By eliminating the protecting group scheme and coupling reagents, tandem ligation of multiple free peptide segments in aqueous solutions enhances the scope of protein synthesis and may provide a useful approach for preparing protein biologicals and synthetic vaccines.
Tam
J P
JP
Department of Microbiology and Immunology, Vanderbilt University, Nashville, TN, U.S.A. tamjp@ctrvax.vanderbilt.edu
Yu
Q
Q
Lu
Y A
YA
D000595:Amino Acid Sequence; D054405:Chemokine CCL3; D054407:Chemokine CCL4; D002851:Chromatography, High Pressure Liquid; D019402:Macrophage Inflammatory Proteins / Q000737:chemistry; D008969:Molecular Sequence Data; D010455:Peptides / Q000737:chemistry* / Q000276:immunology; D013004:Somatostatin / Q000737:chemistry; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
D054405:Chemokine CCL3; D054407:Chemokine CCL4; D019402:Macrophage Inflammatory Proteins; D010455:Peptides; D013004:Somatostatin