earticle

영어

Recombinant human growth hormone (hGH) has been used as a therapeutic agent to treat children's growth disorders and adult growth hormone deficiency. hGH has been known to be highly susceptible to rapid proteolysis and has a short half-life of only a few hours in human blood stream. Here we developed an experimental and rational method to select modification sites in hGH polypeptide, where any changes (glycosylation or site-directed mutagenesis) can be applied. First, we selected several putative permissive sites and performed site-specific glycosylations for improved proteolytic stability. hGHs with site-specific mutations were expressed in Saccharomyces cerevisiae, and their glycosylations were confirmed by SDS-PAGE analysis and glycosidase treatment. The activities of glycosylated hGHs were tested using Nb2 cell proliferation bioassay. When compared with the wild-type unglycosylated hGH, the glycosylated hGH did not lose its biological activity. The proteolytic stability of glycosylated hGH was also tested by western blot analysis after treatment of plasmin. As a result, glycosylated hGH showed enhanced stability compared with the wild-type hGH. These results indicate that glycosylation at internal permissive sites was successful to develop protease-resistant hGH.