High level expression of human endostatin in Pichia pastoris using a synthetic gene construct.
Endostatin, a 20-kDa C-terminal fragment derived from type XVIII collagen, is a potent angiogenesis inhibitor and an antitumor factor. To improve the production of recombinant human endostatin on increasing demand in clinical practice, we constructed an artificial gene encoding its mature peptide sequence in human collagen XVIII. The synthetic gene consisted of 20 codons in preference in methylotropic yeast-Pichia pastoris and was cloned into expression vector pPICZalphaA; and the recombinant protein was expressed in P. pastoris strain SMD1168 and purified to near homogeneity using heparin affinity chromatography. The amount of expressed recombinant protein in cultural media using described strategy was 80 mg/l in shake flask cultivation and 435 mg/l in high-density bioreactor fermentation. Methylthiazolium assay demonstrated that human endostatin expressed in P. pastoris using artificial synthetic gene of preference in P. pastoris was able to inhibit the acidic fibroblast growth factor-induced proliferation of endothelial cells in vitro.
Su
Zhijian
Z
School of Pharmaceutical Science, Wenzhou Medical College, Wenzhou, 325035, China.
Wu
Xiaoping
X
Feng
Ya
Y
Ding
Changcai
C
Xiao
Yechen
Y
Cai
Lu
L
Feng
Wenke
W
Li
Xiaokun
X
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D015153:Blotting, Western; D049109:Cell Proliferation / Q000187:drug effects; D002478:Cells, Cultured; D004305:Dose-Response Relationship, Drug; D004591:Electrophoresis, Polyacrylamide Gel; D043169:Endostatins / Q000235:genetics* / Q000378:metabolism / Q000494:pharmacology; D042783:Endothelial Cells / Q000166:cytology / Q000187:drug effects; D005813:Genes, Synthetic / Q000235:genetics*; D006801:Humans; D051379:Mice; D008969:Molecular Sequence Data; D010843:Pichia / Q000235:genetics* / Q000378:metabolism; D011994:Recombinant Proteins / Q000302:isolation & purification / Q000378:metabolism / Q000494:pharmacology
D043169:Endostatins; D011994:Recombinant Proteins