A Convenient Method for Solubilization and Refolding of Recombinant Proteins: An Experience From Recombinant Mouse Transforming Growth Factor-β 1

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Maleki Fahimeh; Mashayekhi Kazem; Moghadam Malihe; Badiee Kheirabadi Seyedeh Elham; MOUSAVI MOHAMMAD JAVAD; SANKIAN MOJTABA

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Journal: Research in Molecular Medicine Year:2020 | Volume:8 | Issue:1 Page(s): 9-15

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Title

A Convenient Method for Solubilization and Refolding of Recombinant Proteins: An Experience From Recombinant Mouse Transforming Growth Factor-β 1

Author(s)

Keywords

Inclusion bodies

mouse Transforming growth factor-beta 1

Protein expression

Refolding protein

Abstract

Background: The production of recombinant proteins in Escherichia coli (E. coli) is one of the most valuable achievements in biotechnology having many therapeutic and diagnostic applications; however, the aggregation and misfolding of proteins leading to the formation of insoluble Inclusion bodies is a disruptive factor in this process. Various solubilization and refolding methods could be used to improve recombinant protein conformation. In this study, we applied a dilution method with refolding buffer to produce a native form of soluble immature mouse Transforming Growth FactorBeta 1 (TGF-β 1). Materials and Methods: The TGF-β 1 complementary DNA (cDNA) which encodes the protein without the signal peptide was cloned into the pET21-b (+) vector. The target protein was expressed by the transformation of E. coli BL21 cells with the plasmid. The resulting Inclusion bodies were diluted in lysis buffer and solubilized in refolding buffer to make a protein with native structure. The protein quantification was performed by using Bicinchoninic Acid Assay (BCA). Results: Following Polymerase Chain Reaction (PCR) of the recombinant plasmid with T7 primers, electrophoresis and sequencing of the amplified product indicated 100% target sequence identity with the murine TGF-β 1 gene. Finally, the protein solubility and immuno-reactivity were confirmed a 44 kDa protein which conducted with the anti-TGF-β 1-specific polyclonal antibody by western blot. The protein quantification with the BCA method showed 30 µ g/mL concentration. Conclusion: The dilution method and refolding buffer used in this study could effectively convert aggregated immature mouse TGF-β 1 to a soluble and immuno-reactive form.

Cites

Cloning, Expression, and Purification of Recombinant Mouse Interferon-γ

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APA: Copy

Maleki, Fahimeh, Mashayekhi, Kazem, Moghadam, Malihe, Badiee Kheirabadi, Seyedeh Elham, MOUSAVI, MOHAMMAD JAVAD, & SANKIAN, MOJTABA. (2020). A Convenient Method for Solubilization and Refolding of Recombinant Proteins: An Experience From Recombinant Mouse Transforming Growth Factor-β 1. RESEARCH IN MOLECULAR MEDICINE, 8(1), 9-15. SID. https://sid.ir/paper/704267/en

Vancouver: Copy

Maleki Fahimeh, Mashayekhi Kazem, Moghadam Malihe, Badiee Kheirabadi Seyedeh Elham, MOUSAVI MOHAMMAD JAVAD, SANKIAN MOJTABA. A Convenient Method for Solubilization and Refolding of Recombinant Proteins: An Experience From Recombinant Mouse Transforming Growth Factor-β 1. RESEARCH IN MOLECULAR MEDICINE[Internet]. 2020;8(1):9-15. Available from: https://sid.ir/paper/704267/en

IEEE: Copy

Fahimeh Maleki, Kazem Mashayekhi, Malihe Moghadam, Seyedeh Elham Badiee Kheirabadi, MOHAMMAD JAVAD MOUSAVI, and MOJTABA SANKIAN, “A Convenient Method for Solubilization and Refolding of Recombinant Proteins: An Experience From Recombinant Mouse Transforming Growth Factor-β 1,” RESEARCH IN MOLECULAR MEDICINE, vol. 8, no. 1, pp. 9–15, 2020, [Online]. Available: https://sid.ir/paper/704267/en

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