| چکیده انگلیسی مقاله |
Background: The production of recombinant proteins in Escherichia coli is one of the most valuable achievements in biotechnology, with many therapeutic and diagnostic applications; however, the aggregation and misfolding of proteins that result in the formation of insoluble inclusion bodies is a disruptive factor in this process. Various solubilization and refolding methods can be used to improve recombinant protein conformation. In this study, we applied a dilution method with a refolding buffer to produce a native form of soluble immature mouse TGF-β1. Methods: The TGF-β1 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 solubilized and then diluted in the refolding buffer to make a protein with native structure. Results: Following PCR of the recombinant plasmid with T7 primers, electrophoresis and sequencing of the amplified product indicated 100% identity of the target sequence with the murine TGF-β1 gene. Finally, the protein solubility and immuno-reactivity were confirmed a 44 kDa protein which conducted with the anti-TGF-β1-specific polyclonal antibody on a western blot. Conclusion: Our dilution method and refolding buffer effectively converted aggregated immature mouse TGF-β1 to a soluble and immuno-reactive form. |
| نویسندگان مقاله |
| Fahimeh Maleki
Immuno-Biochemistry lab, Immunology Research Center, Buali Research Institute, School of Medicine, Mashhad University of Medical Sciences, Mashhad, Iran
| Kazem Mashayekhi
Immuno-Biochemistry lab, Immunology Research Center, Buali Research Institute, School of Medicine, Mashhad University of Medical Sciences, Mashhad, Iran
| Seyedeh Elham Badiee Kheirabadi
Immuno-Biochemistry lab, Immunology Research Center, Buali Research Institute, School of Medicine, Mashhad University of Medical Sciences, Mashhad, Iran
| Mohammad Javad Mousavi
Immunology Department, School of Medicine, Tehran University of Medical Sciences, Tehran, Iran
| Mojtaba Sankian
Immuno-Biochemistry lab, Immunology Research Center, Buali Research Institute, School of Medicine, Mashhad University of Medical Sciences, Mashhad, Iran
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