title: N-terminal modification increases the stability of the recombinant human endostatin in vitro.
abstract: Endostar, approved for the treatment of non-small-cell lung cancer by the State Food and Drug Administration in China, is a derivative of human endostatin that is modified with an additional metal-chelating sequence (MGGSHHHHH) at the N-terminus. This modification contributes to an additional zinc-binding site in the endostatin sequence. In the present study, zinc-binding and zinc-free endostar were compared to further characterize their biochemical and structural properties. Thermally induced denaturation was determined by monitoring changes in fluorescence emission spectra. The data indicated that zinc binding significantly increased the transition temperature of endostar and contributed to a reversible change in protein conformation after recooling. Proteolysis assays demonstrated that the modified protein binding with zinc ions can stabilize the N-terminus and the C-terminus of endostar when treated with trypsin, chymotrypsin and carboxypeptidase A and B. Western-blot analyses using anti-His6 antibody confirmed that the major cleaved fragments of endostar were in the N-terminus when treated with trypsin and chymotrypsin. In the proliferation assay with human umbilical-vein endothelial cells, the zinc-binding and zinc-free endostar samples with extra zinc-binding sites displayed similar inhibiting activities.
authors.affiliation: Jiangsu Simcere Pharmaceutical R&D Co., Ltd, 699-18 Xuan Wu Avenue, Nanjing 210042, People's Republic of China.
authors.forename: Li-Ping
authors.initials: LP
authors.lastname: Jiang
authors.forename: Chang
authors.initials: C
authors.lastname: Zou
authors.forename: Xue
authors.initials: X
authors.lastname: Yuan
authors.forename: Wei
authors.initials: W
authors.lastname: Luo
authors.forename: Yong
authors.initials: Y
authors.lastname: Wen
authors.forename: Yali
authors.initials: Y
authors.lastname: Chen
mesh_terms: D001059:Apoproteins; D015153:Blotting, Western; D049109:Cell Proliferation / Q000187:drug effects; D002478:Cells, Cultured; D043169:Endostatins / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism / Q000494:pharmacology; D042783:Endothelial Cells; D006801:Humans; D055550:Protein Stability; D011994:Recombinant Proteins; D012697:Serine Endopeptidases / Q000378:metabolism; D013050:Spectrometry, Fluorescence; D014364:Tryptophan / Q000737:chemistry; D014471:Umbilical Veins / Q000166:cytology; D015032:Zinc / Q000378:metabolism
chemical_list: D001059:Apoproteins; D043169:Endostatins; D011994:Recombinant Proteins; D014364:Tryptophan; D012697:Serine Endopeptidases; C522911:endostar protein; D015032:Zinc