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

IF 1.6 Q4 BIOCHEMISTRY & MOLECULAR BIOLOGY Reports of Biochemistry and Molecular Biology Pub Date : 2023-04-01 DOI:10.52547/rbmb.12.1.27
Masoud Hashemzaei, Manica Negahdaripour, Reza Heidari, Mohammad Bagher Ghoshoon
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引用次数: 1

Abstract

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 BL21. For analysis of the effect of the pelB signal peptide, pET-22b (+) and pET-15b plasmids were used. The probability of signal peptide cleavage and pathway was predicted by using the SignalP 5.0 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.

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Romiplostim的蛋白表达、纯化及其在大肠杆菌中的分泌产物分析。
背景:罗米普洛司汀是一种血小板生成素受体激动剂,被批准用于治疗免疫性血小板减少症。它是通过重组DNA技术在大肠杆菌中产生的。许多研究人员已经通过使用信号肽序列研究了重组蛋白在大肠杆菌中的周质或细胞外生产,因为它与细胞内生产相比具有优势。在本研究中,分析了pelB信号肽对Romiplostim产生的影响。方法:对Romiplostim的核苷酸序列进行密码子优化,使其在大肠杆菌BL21中表达。为了分析pelB信号肽的作用,使用pET-22b(+)和pET-15b质粒。利用SignalP5.0程序预测了信号肽裂解的可能性和途径,并分析了重组蛋白的表达、纯化和生物活性。结果:计算机分析预测了pelB信号肽的正确切割。然而,实验结果显示,与该信号肽融合的蛋白质在细胞内积聚,在周质或细胞外空间中没有任何可检测的蛋白质带。与对照组相比,不含信号肽的纯化蛋白质的体内实验显示血小板显著增加。结论:Romiplostim在含有或不含有信号肽的大肠杆菌中均有表达。最新的一个显示出合适的体内生物活性。尽管有计算机预测的结果,但在实验条件下,pelB信号肽不能将蛋白质转运到周质或细胞外环境中。尝试不同的信号肽和更多的计算机分析可能有助于Romiplostim蛋白的有效分泌。
本文章由计算机程序翻译,如有差异,请以英文原文为准。
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来源期刊
Reports of Biochemistry and Molecular Biology
Reports of Biochemistry and Molecular Biology BIOCHEMISTRY & MOLECULAR BIOLOGY-
CiteScore
2.80
自引率
23.50%
发文量
60
审稿时长
10 weeks
期刊介绍: The Reports of Biochemistry & Molecular Biology (RBMB) is the official journal of the Varastegan Institute for Medical Sciences and is dedicated to furthering international exchange of medical and biomedical science experience and opinion and a platform for worldwide dissemination. The RBMB is a medical journal that gives special emphasis to biochemical research and molecular biology studies. The Journal invites original and review articles, short communications, reports on experiments and clinical cases, and case reports containing new insights into any aspect of biochemistry and molecular biology that are not published or being considered for publication elsewhere. Publications are accepted in the form of reports of original research, brief communications, case reports, structured reviews, editorials, commentaries, views and perspectives, letters to authors, book reviews, resources, news, and event agenda.
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