Expression, purification and characterization of soluble human thrombopoietin receptor from Escherichia coli

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• 作者：Heungrok Park ; Hana Im ; Young Jun Kang ; Myeong-Hee Yu and Hyo Jeong Hong
• 关键词：inclusion bodies ; protein purification ; receptor domain ; thrombopoietin
• 刊名：Biotechnology Letters
• 出版年：2000
• 出版时间：2000
• 年：2000
• 卷：22
• 期：20
• 页码：1611-1617
• 全文大小：78 KB
• 刊物类别：Biomedical and Life Sciences
• 刊物主题：Life Sciences
  Microbiology
  Biotechnology
  Applied Microbiology
  Biochemistry
  
• 出版者：Springer Netherlands
• ISSN：1573-6776

文摘

The extracellular domain (edMpl) of human thrombopoietin (TPO) receptor, c-Mpl was expressed in Escherichia coli by changing some nucleotides before and after the translation initiation codon. The mutations increased the expression by approx. 15-fold. The inclusion bodies were solubilized in 8 M guanidine-HCl under reducing conditions and refolded using a glutathione-redox system. The monomeric form of edMpl was purified to near homogeneity by two successive steps of ion-exchange chromatography using DEAE-Sephacel and Mono Q columns. The purified monomeric edMpl inhibited the TPO-dependent cell proliferation, suggesting that it was binding to TPO. Also, antisera raised against the edMpl bound specifically to the soluble receptor secreted by mammalian cells.