DOMAIN LIGATION STRATEGY TO ENGINEER PROTEINS WITH UNUSUAL ARCHITECTURES

摘要

A novel method of chemical ligation of peptide segments requires no protecting groups and no activation of the C-α carboxyl groups. The method consists of four steps: (1) aldehyde initiation in which a masked glycolaldehyde ester is linked to the carboxylic group of an unprotected peptide by enzymatic reaction; (2) ring formation - the regenerated aldehyde reacts with the N-α amine of the second unprotected peptide; (3) rearrangement where O-acyl linkage is transferred to N-acyl linkage to form a peptide bond at higher pH; (4) reconversion to the natural amino acids if necessary. This invention also relates to a method for ligating unprotected peptide, proteins or nonpeptide segments to give therapeutic products and synthetic vaccines with linear, circularized, or branched backbone structures. The method embraces the discovery that when a weakly basic nucleophile and aldehyde are used as a reacting pair for the ligation of two segments, the initial covalent formation of thiazolidine, oxazolidine, hydrozone and oxime bonds is highly specific and may undergo a subsequent intramolecular O to N-acyl rearrangement step which results in the formation of amide bond. Weak bases on a peptide segment are those that contain 1,2- or 1,3-amino thiol or alcohol or those that contain an electronic withdrawing group to the amine such as hydrazide, oxime, phenylhydrazine. Unprotected peptides containing amino terminal residues of cysteine, serine, or threonine can therefore be served as a weak base or as a masked aldehyde which can be generated by oxidation. This invention also relates to a method using the same concept of weak base-aldehyde ligation for site-specific modification of peptides or proteins by lipidation and pegylation. More particularly, the invention relates to the modification of the protein gp120 derived from the human immunodeficiency virus-1 at the amino terminus to contain one or more lipid side chains (lipidation) to increase its efficacy for vaccine and the modificacition of cytokine interleukin-2 by polyethylene glycol (PEG, pegylation) to increase its stability.