We developed a new semisynthetic method of homogenous glycoprotein. We prepared long peptide segments and a short glycopeptide by E. coli expression system and chemical synthesis, respectively. In order to obtain a sufficient amount of glycopeptide substrate, we studied an improved chemical method. In addition, we also studied an efficient thioesterification of peptide prepared in E.coli. These improved methods efficiently gave full length glycoprotein.

Sufficient quantities of homogeneous glycoproteins are often not readily available to facilitate studies of glycan structure-function relationship. We synthesized a single glycoform by the semisynthetic method, which involves recombinant expression and chemical synthesis. We selected interferon-β as the target and designed four segments coupling strategy. A short segment C containing a glycan were synthesized by an improved chemical method, while other segments A, B and D were prepared by E.coli expression system. We developed a thioesterification method for E. Coli expressed peptide via cys-cyanylation followed by hydrazinolysis condition. All segments were successfully ligated and we obtained a full length interferon-β glycosyl polypeptide.