Expression,refolding, and characterization of recombinant thrombopoietin/stem cell factor fusion protein in <Emphasis Type="Italic">Escherichia coli</Emphasis> |
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| Authors: | Yuhui Zang Xu Zhang Xiaoling Jiang Haoran Li Jie Zhu Chi Zhang Wei Peng Junchuan Qin |
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| Affiliation: | (1) State Key Laboratory of Pharmaceutical Biotechnology, School of Life Science, Nanjing University, Nanjing, 210093, People’s Republic of China;(2) Institute of Agrobiological Genetics and Physiology, Jiangsu Academy of Agricultural Sciences, Nanjing, 210014, People’s Republic of China |
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| Abstract: | Thrombopoietin/stem cell factor (TPO/SCF) is a novel fusion protein that combines the complementary biological effects of
TPO and SCF into a single molecule. In this study, TPO/SCF gene was cloned into pET32a and expressed as a thioredoxin (Trx)
fusion protein with a C-terminal 6His-tag in Escherichia coli BL21(DE3) under the control of T7 promoter. Trx-TPO/SCF protein approximately accounted for 20% of the total bacterial proteins
and was found to accumulate in inclusion bodies. Inclusion bodies were separated from cellular debris, washed with buffer
containing 2 M urea, and solubilized with 8 M urea. The refolding of Trx-TPO/SCF was then carried out by an on-column method.
Soluble Trx-TPO/SCF was characterized for its dose-dependent effects on promoting cells proliferation in both TF1 and Mo7e
cell lines. rhTPO/SCF was released by thrombin digestion and further purified by Ni2+ affinity chromatography. Western blot analysis confirmed the identities of Trx-TPO/SCF and rhTPO/SCF. |
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| Keywords: | TPO/SCF Megakaryopoiesis Bacterial expression Inclusion body Refolding |
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