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Expression of Tumor Necrosis Factor-Related Apoptosis- Inducing Ligand Conjugated to the scFV Region of Anti-CD20 Antibody Using a Small Ubiquitin-related Modifier
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Mahsa Rasekhian1    , MARYAM Pourjalili2 , Omid Tavallaei 3 |
1- Pharmaceutical Sciences Research Center, Health Institute, Kermanshah University of Medical Sciences, Kermanshah, Iran
2- students research committee, Kermanshah university of medical sciences, Kermanshah, Iran
3- Pharmaceutical Sciences Research Center, Health Institute, Kermanshah University of Medical Sciences, Kermanshah, Iran , o.tavallaei61@gmail.com |
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Abstract: (884 Views) |
Background and Aim: Conventional treatments have shown little success in cancer treatment. Using antibody fragments conjugated with specific proteins and targeting specific receptors is a new strategy for producing anticancer drugs. Despite the advantages of prokaryotic hosts, protein expression in the form of inclusion bodies (cytoplasmic) has been challenging and leads to subsequent difficulties. Production of proteins as soluble forms will significantly help to solve this problem.
The primary purpose of this study was to clone and express Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) conjugated with anti CD20 single chain fragment variable (scFV) region of anti-CD20 antibody as a soluble protein using a small ubiquitin-related modifier (SUMO) fusion protein system in E. coli as a new method for optimal production of anticancer drugs.
Materials and Methods: Appropriate primers for a previously designed DNA sequence encoding the fusion protein were used to amplify the fragment. The amplified fragment was sub-cloned downstream of the SUMO tag in pSUMO vector. Once confirmed by standard methods, the recombinant plasmid was transformed into E. coli strain BL21 (DE3). Expression was induced by isopropyl β- d-1-thiogalactopyranoside. The expressed proteins were assessed by SDS-PAGE and then isolated by affinity chromatography. Western blotting was used to confirm protein expression.
Results: The results showed the DNA of the recombinant fusion protein was sub-cloned correctly, and the relevant analyses indicated that the expression process was successful. The recombinant fusion protein was confirmed by appropriate analysis.
Conclusion: The production of recombinant proteins in a soluble state in the prokaryotic host E. coli can reduce the costs of the downstream process. Production of soluble antiCD20 scFV-TRAIL fusion protein can be considered a proper method to produce recombinant protein in a soluble state in the E. coli system and can decrease the cost of downstream processes. |
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| Keywords: Fusion protein, Expression, Purification, Recombinant, Single chain fragment variable, Tumor necrosis factor-related apoptosis-inducing ligand |
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Full-Text [PDF 501 kb]
(284 Downloads)
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Type of Study: Original Research |
Subject:
Biotechnology
Received: 2022/04/18 | Accepted: 2022/08/16 | Published: 2023/09/27
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Ethics code: IR.KUMS.REC.1397.112
rasekhian M, pourjalili M, tavallaei O. Expression of Tumor Necrosis Factor-Related Apoptosis- Inducing Ligand Conjugated to the scFV Region of Anti-CD20 Antibody Using a Small Ubiquitin-related Modifier. SJKU 2023; 28 (4) :12-23 URL: http://sjku.muk.ac.ir/article-1-7310-en.html
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