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مقالات فارسیISIکنفرانسهاژورنالها

Protein Expression and Purification of Romiplostim and Analysis of Its Secretory Production Using an In Silico Investigated Signal Peptide in E. Coli

محل انتشار: مجله گزارش های بیوشیمی و زیست شناسی مولکولی، دوره: 12، شماره: 1
سال انتشار: 1402
نوع سند: مقاله ژورنالی
زبان: انگلیسی
مشاهده: 68

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https://civilica.com/doc/1736000

شناسه ملی سند علمی:

JR_RBMB-12-1_003

تاریخ نمایه سازی: 31 مرداد 1402

چکیده مقاله:

Background: Romiplostim is a thrombopoietin receptor agonist approved for the treatment of immune thrombocytopenia. It is produced by recombinant DNA technology in Escherichia coli. Many researchers have studied the periplasmic or extracellular production of recombinant proteins in E. coli by using signal peptide sequences due to its advantages compared to intracellular production. In this study, the effect of the pelB signal peptide on Romiplostim production was analyzed. Methods: The nucleotide sequence of Romiplostim was codon optimized for expression in E. coli BL۲۱. For analysis of the effect of the pelB signal peptide, pET-۲۲b (+) and pET-۱۵b plasmids were used. The probability of signal peptide cleavage and pathway was predicted by using the SignalP ۵.۰ program, and expression, purification, and biological activity of the recombinant protein were analyzed. Results: In-silico analysis predicted the correct cleavage of the pelB signal peptide. However, the experimental results showed intracellular accumulation of the protein in fusion with this signal peptide without any detectable protein band in periplasmic or extracellular spaces. The in-vivo experiment of purified protein without signal peptide exhibited a significant increment in platelets compared to the control group. Conclusions: Romiplostim was expressed in E. coli with and without signal peptide. The latest one showed suitable in-vivo bioactivity. Despite the results of in-silico prediction, the pelB signal peptide could not transport the protein into the periplasm or extracellular environment in the experimental condition. Trying different signal peptides and more in-silico analysis might be helpful for the efficient secretion of the Romiplostim protein.

کلیدواژه ها:

E. coli ، Romiplostim ، Secretory production ، Signal peptide ، Thrombocytopenia.

نویسندگان

Masoud Hashemzaei

Pharmaceutical Sciences Research Center, Shiraz University of Medical Sciences, Shiraz, Iran & Department of Pharmaceutical Biotechnology, School of Pharmacy, Shiraz University of Medical Sciences, Shiraz, Iran.

Manica Negahdaripour

Pharmaceutical Sciences Research Center, Shiraz University of Medical Sciences, Shiraz, Iran & Department of Pharmaceutical Biotechnology, School of Pharmacy, Shiraz University of Medical Sciences, Shiraz, Iran.

Reza Heidari

Pharmaceutical Sciences Research Center, Shiraz University of Medical Sciences, Shiraz, Iran.

Mohammad Bagher Ghoshoon*

Pharmaceutical Sciences Research Center, Shiraz University of Medical Sciences, Shiraz, Ira & Biotechnology Research Center, Shiraz University of Medical Sciences, Shiraz, Iran.

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