Abstract
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.
This is a preview of subscription content, log in via an institution to check access.
Similar content being viewed by others
References
Alexander WS, Metcalf D, Dunn AR (1995) Point mutations within a dimer interface homology domain of c-Mpl induce constitutive receptor activity and tumorigenicity. EMBO J. 14: 5569-5578.
Bartley TD, Bogenberger J, Hunt P, Li YS, Lu HS, Martin F, Chang MS, Samal B, Nichol JL, Swift S (1994) Identification and cloning of a magakaryocyte growth and development factor that is a ligand for the cytokine receptor Mpl. Cell 77: 1117-1124.
Deane CM, Kroemer RT, Richards WG (1997) A structural model of the human thrombopoietin receptor complex. J. Mol. Graph. Model 15: 170-178.
de Sauvage FJ, Hass PE, Spencer SD, Malloy BE, Gurney AL, Spencer SA, Darbonne WC, Henzel WJ, Wong SC, Kuang WJ (1994) Stimulation of megakaryocytopoiesis and thrombopoiesis by the c-Mpl ligand. Nature 369: 533-538.
Habeeb AFSA (1972) Reaction of protein sulfhydryl groups with Ellman's reagent. Meth. Enzymol. 25: 457-464.
Hui A, Hayflick J, Dinkelspiel K, de Boer HA (1984) Mutagenesis of the three bases preceding the start codon of the betagalactosidase mRNA and its effect on translation in Escherichia coli. EMBO J. 3: 623-629.
Kang YJ, Kim YS, Hur H, Kim HS, Hong HJ (1999) Efficient expression and characterization of hepatitis B virus preS2 antigen in Escherichia coli. Biotechnol. Lett. 21: 375-380.
Kaushansky K, Lok S, Holly RD, Broudy VC, Lin N, Bailey MC, Forstrom JW, Buddle MM, Oort PJ, Hagen FS (1994) Promotion of megakaryocyte progenitor expansion and differentiation by the c-Mpl ligand thrombopoietin. Nature 369: 568-571.
Lok S, Kaushansky K, Holly RD, Kuijper JL, Lofton-Day CE, Oort PJ, Grant FJ, Heipel MD, Burkhead SK, Kramer JM (1994) Cloning and expression of murine thrombopoietin cDNA and stimulation of platelet production in vivo. Nature 369: 565-568.
Looman AC, Bodlaender J, Comstock LJ, Eaton D, Jhurani P, de Boer HA, van Knippenberg PH (1987) Influence of the codon following the AUG initiation codon on the expression of a modified lacZ gene in Escherichia coli. EMBO J. 6: 2489-2492.
Park H, Hong HJ (1997) Development of an in vitrobioassay system for human thrombopoietin by constructing a recombinant murine cell line expressing human thrombopoietin receptor. Mol. Cells 7: 699-704.
Park H, Park SS, Jin EH, Song J-S, Ryu S-E, Yu M-H, Hong HJ (1998) Identification of functionally important residues of human thrombopoietin. J. Biol. Chem. 273: 256-261.
Vigon I, Mornon JP, Cocault L, Mitjavila MT, Tambourin P, Gisselbrecht S, Souyri M (1992) Molecular cloning and characterization of MPL, the human homolog of the v-mpl oncogene: identification of a member of the hematopoietic growth factor receptor superfamily. Proc. Natl. Acad. Sci. USA 89: 5640-5644.
Author information
Authors and Affiliations
Rights and permissions
About this article
Cite this article
Park, H., Im, H., Kang, Y.J. et al. Expression, purification and characterization of soluble human thrombopoietin receptor from Escherichia coli. Biotechnology Letters 22, 1611–1617 (2000). https://doi.org/10.1023/A:1005672824663
Issue Date:
DOI: https://doi.org/10.1023/A:1005672824663