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N‐terminal modification increases the stability of the recombinant human endostatin in vitro
Author(s) -
Jiang LiPing,
Zou Chang,
Yuan Xue,
Luo Wei,
Wen Yong,
Chen Yali
Publication year - 2009
Publication title -
biotechnology and applied biochemistry
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.468
H-Index - 70
eISSN - 1470-8744
pISSN - 0885-4513
DOI - 10.1042/ba20090063
Subject(s) - zinc , endostatin , trypsin , chymotrypsin , carboxypeptidase a , chemistry , microbiology and biotechnology , biochemistry , western blot , recombinant dna , proteolysis , in vitro , denaturation (fissile materials) , binding site , biology , carboxypeptidase , enzyme , angiogenesis , nuclear chemistry , organic chemistry , cancer research , gene
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 (MGGS) 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‐His 6 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.