High level expression of human endostatin in Pichia pastoris using a synthetic gene construct

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• 作者：Zhijian Su ; Xiaoping Wu ; Ya Feng ; Changcai Ding ; Yechen Xiao ; Lu Cai ; Wenke Feng and Xiaokun Li
• 关键词：Endostatin ; Pichia pastoris ; Artificial synthetic gene ; High ; density fermentation
• 刊名：Applied Microbiology and Biotechnology
• 出版年：2007
• 出版时间：January, 2007
• 年：2007
• 卷：73
• 期：6
• 页码：1355-1362
• 全文大小：278 KB
• 刊物类别：Chemistry and Materials Science
• 刊物主题：Chemistry
  Biotechnology
  Microbiology
  Microbial Genetics and Genomics
  
• 出版者：Springer Berlin / Heidelberg
• ISSN：1432-0614

文摘

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 pPICZαA; 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.