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|Title: ||Expression of the recombinant human thrombopoietin receptor (rhTPOR) in escherichia coli and mammalian cells|
|Authors: ||Ng, Kit-fong|
|Issue Date: ||1999 |
|Abstract: ||Thrombopoietin receptor (MPL or TPOR) is a member of the cytokine receptor superfamily expressed primarily on hematopoietic cells. TPOR plays an important role in regulating platelet production. Due to its low expression level in human tissue, studies on the biochemical and biophysical characteristics of TPOR have been limited. The objective of this study is to express a reasonable amount of TPOR in E. coli and mammalian cells to facilitate such studies.
E. coli was chosen to produce TPOR because of its ease to manipulate and fast growth rate. A cDNA encoding the extracellular domain of human TPOR (TPOR-EN) was cloned from fetal liver mRNA, sequenced and linked to a bacterial expression vector, pET 30a. The TPOR-EN cDNA was over-expressed in E. coli as inclusion body under optimized conditions. The expressed protein was present as inclusion body. The latter was solubilized in urea and was purified by immobilized metal affinity chromatography under denaturing conditions. The purified TPOR-EN was refolded by gel filtration chromatography and was shown to have a predominantly β sheet structure by Far-UVcircular dichroism. In addition, TPOR-EN was shown to bind to its ligand, a commercial rh TPO, in a dose-dependent manner in surface plasmon resonance experiment.
In addition, a mammalian cell line expressing the full length human TPOR was established. A full length TPOR cDNA was synthesized from human fetal liver mRNA, was sequenced and was cloned into a pcDNA 3.1 mammalian expression vector. The expression plasmid was used to transfect an IL-3 dependent cell line, 32D Clone 3 cells. Characterization of the transfected cells were perforrned by cultivating the transformants in the medium supplemented with exogenous rhTP0, rmIL-3 or rmIL-6. The result shown that rhTP0 and rmIL-3 but not rmIL-6 could support the survival and growth of the transformants, indicating that TPOR protein was expressed. Attempts to establish a homogeneous cell clone are in progress.|
|Description: ||Thesis (M.Phil.)--Hong Kong University of Science and Technology, 1999|
xxi, 109 leaves : ill. (some col.) ; 30 cm
HKUST Call Number: Thesis BIOL 1999 Ng
|Appears in Collections:||BIOL Master Theses|
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