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:: Volume 11, Issue 1 (March 2019 2019) ::
IJBC 2019, 11(1): 6-12 Back to browse issues page
Optimized Cytoplasmic Expression of Water Soluble Human Thrombopoietin in Modified Bacterial Strain
Mohammad Mouradi, Kamran Atarodi, Mahshid Mohammadipour, Kamran Mousavi Hosseini *
Blood Transfusion Research Center, High Institute for Research and Education in Transfusion Medicine
Abstract:   (455 Views)
Background: Thrombopoietin is a glycoprotein produced by liver and kidney which is responsible for regulating the platelet production. Thrombopoietin is a key ligand with impact on regulating the self-renewal of Hematopoietic stem cells and the regulation of Megakaryocytes progenitors. Previous studies have indicated that only N-terminal domain of this protein has receptor promoting ability. The goal of this study was to express the recombinant form of the soluble Thrombopoietin by using Rosetta gami strain of Escherichia coli as the expression host and pET vectors system to produce this cytokine in a cost-effective manner.
Methods: After isolating the genomic sequence of Thrombopoietin from HepG2 cDNA, it was inserted into the pET32 vector and colonized by DH5α. Then, recombinant plasmids were transformed into Rosetta gami to express the recombinant protein. Expression was analyzed through SDS-PAGE and western blotting assays. Finally, the optimized time for induction and the suitable concentrations of IPTG were determined. 
Results: DNA sequencing of recombinant plasmid proved that the sequence was indicative of variant 2 of Thrombopoietin. SDS-PAGE and western blotting showed a 36-kDa protein band which was compatible with the inserted sequence. Optimized time for expression was 6 h and optimized concentration of IPTG was 0.1 mM.
Conclusion: Although different methods are available to produce recombinant Thrombopoietin, low yield and costly setup are major obstacles in the production of Thrombopoietin. Also the products of usual bacterial systems are mostly inclusion bodies which is due to lacking disulfide bond formation. In this study, mutant strain of E. coli was used to provide a high level of water-soluble Thrombopoietin expression. 
Keywords: Thrombopoietin, Recombinant Protein, Escherichia coli, Blood Platelets
Full-Text [PDF 1201 kb]   (184 Downloads)    
: Research | Subject: Adults Hematology & Oncology
Received: 2018/12/4 | Accepted: 2019/03/12
* Corresponding Author Address: Blood Transfusion Research Center, High Institute for Research and Education in Transfusion Medicine, Next to the Milad Tower, Tehran, Iran.
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Mouradi M, Atarodi K, Mohammadipour M, Mousavi Hosseini K. Optimized Cytoplasmic Expression of Water Soluble Human Thrombopoietin in Modified Bacterial Strain. IJBC. 2019; 11 (1) :6-12
URL: http://ijbc.ir/article-1-856-en.html


Volume 11, Issue 1 (March 2019 2019) Back to browse issues page
مجله ی خون و سرطان ایران Iranian Journal of Blood and Cancer
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