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'); Reports of Biochemistry and Molecular Biology rbmb.net Basic Sciences http://rbmb.net 1 admin 2322-3480 2322-3480 10.61882/rbmb en jalali 1402 1 1 gregorian 2023 4 1 12 1 online 1 fulltext
en Protein Expression and Purification of Romiplostim and Analysis of Its Secretory Production Using an In Silico Investigated Signal Peptide in E. Coli زیست شناسی ملکولی Molecular Biology مقالات اصلی Original Article <div style="text-align: justify;"><span style="text-justify:kashida"><span style="text-kashida:0%"><span style="line-height:normal"><span style="tab-stops:396.55pt"><b><i><span style="font-size:12.0pt"><span new="" roman="" style="font-family:" times=""><span style="letter-spacing:-.3pt">Background:</span></span></span></i></b><b><i> </i></b><span style="font-size:12.0pt"><span new="" roman="" style="font-family:" times=""><span style="letter-spacing:-.3pt">Romiplostim is a thrombopoietin receptor agonist approved for the treatment of immune thrombocytopenia. It is produced by recombinant DNA technology in <i>Escherichia coli</i>. Many researchers have studied the periplasmic or extracellular production of recombinant proteins in&nbsp;<i>E. coli</i> 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.</span></span></span><br> <span lang="IN" style="font-size:12.0pt"><span new="" roman="" style="font-family:" times=""><span style="letter-spacing:-.3pt"></span></span></span><span lang="IN" style="font-size:12.0pt"><span new="" roman="" style="font-family:" times=""><span style="letter-spacing:-.3pt"></span></span></span><span lang="IN" style="font-size:12.0pt"><span new="" roman="" style="font-family:" times=""><span style="letter-spacing:-.3pt"></span></span></span></span></span></span></span><br> <span style="text-justify:kashida"><span style="text-kashida:0%"><span style="line-height:normal"><span style="tab-stops:396.55pt"><b><i><span style="font-size:12.0pt"><span new="" roman="" style="font-family:" times=""><span style="letter-spacing:-.3pt">Methods:</span></span></span></i></b> <span style="font-size:12.0pt"><span new="" roman="" style="font-family:" times="">The nucleotide sequence of Romiplostim was codon optimized for expression in <i>E. coli</i> 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.</span></span></span></span></span></span><br> <br> <span style="font-size:10pt"><span style="text-justify:inter-ideograph"><span style="line-height:normal"><span style="text-autospace:none"><span style="font-family:Calibri,sans-serif"><b><i><span style="font-size:12.0pt"><span new="" roman="" style="font-family:" times=""><span style="letter-spacing:-.3pt">Results:</span></span></span></i></b> <i><span style="font-size:12.0pt"><span new="" roman="" style="font-family:" times="">In-silico </span></span></i><span style="font-size:12.0pt"><span new="" roman="" style="font-family:" times="">analysis predicted the correct cleavage of the&nbsp;</span></span></span></span></span></span></span><span style="font-size:12.0pt"><span style="line-height:110%"><span new="" roman="" style="font-family:" times="">pelB&nbsp;</span></span></span><span style="font-size:10pt"><span style="text-justify:inter-ideograph"><span style="line-height:normal"><span style="text-autospace:none"><span style="font-family:Calibri,sans-serif"><span style="font-size:12.0pt"><span new="" roman="" style="font-family:" times="">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 <i>in-vivo</i> experiment of purified protein without signal peptide exhibited a significant increment in platelets compared to the control group.</span></span></span></span></span></span></span><br> <br> <span style="font-size:10pt"><span style="text-justify:inter-ideograph"><span style="line-height:normal"><span style="text-autospace:none"><span style="font-family:Calibri,sans-serif"><b><i><span style="font-size:12.0pt"><span new="" roman="" style="font-family:" times=""><span style="letter-spacing:-.3pt">Conclusions:</span></span></span></i></b> <span style="font-size:12.0pt"><span new="" roman="" style="font-family:" times=""><span style="letter-spacing:-.3pt">Romiplostim was expressed in <i>E. coli</i> with and without signal peptide. The latest one showed suitable <i>in-vivo</i> bioactivity. Despite the results of <i>in-silico</i> 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 <i>in-silico</i> analysis might be helpful for the efficient secretion of the Romiplostim protein. </span></span></span></span></span></span></span></span></div> E. coli, Romiplostim, Secretory production, Signal peptide, Thrombocytopenia. 27 35 http://rbmb.net/browse.php?a_code=A-10-1182-1&slc_lang=en&sid=1 Masoud Hashemzaei 100319475328460017798 100319475328460017798 No 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 negahdaripour@sums.ac.ir 100319475328460017799 100319475328460017799 Yes 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 100319475328460017800 100319475328460017800 No Pharmaceutical Sciences Research Center, Shiraz University of Medical Sciences, Shiraz, Iran. Mohammad Bagher Ghoshoon* ghoshoonm@sums.ac.ir 100319475328460017801 100319475328460017801 No Pharmaceutical Sciences Research Center, Shiraz University of Medical Sciences, Shiraz, Ira & Biotechnology Research Center, Shiraz University of Medical Sciences, Shiraz, Iran.