en Other Other Original article Original article <div style="text-align: justify;"><b><span style="font-size:10.0pt"><span style="line-height:107%"><span new="" roman="" style="font-family:" times="">Introduction: </span></span></span></b><span style="font-size:10.0pt"><span style="line-height:107%"><span new="" roman="" style="font-family:" times="">The expression of the Cas9 protein in the prokaryotic host <i>Escherichia coli</i> is often associated with the formation of inclusion bodies. This study aimed to express and purify the Cas9 protein in large quantities and soluble form in <i>E. coli</i> BL21 (DE3) for use in the CRISPR/Cas9 system. Due to the large size of Cas9, two solubility-enhancing fusion partners with relatively low molecular weights, initiation factor 2 (IF2) and small ubiquitin-like modifier (SUMO), were utilized. <b>Methods:</b> The <i>IF2</i> and <i>SUMO</i> gene sequences, 480 and 300 bp in length, respectively, were amplified by PCR and cloned into the pET-21a-3xNLS-SpCas9 vector. The expression of both recombinant proteins, with approximate molecular weights of 176 and 166 kDa, was confirmed by SDS-PAGE and Western blotting. Protein purification was performed using Ni-NTA affinity chromatography. <b>Results:</b> The presence of IF2 as a fusion partner increased the percentage of Cas9 detected in the supernatant from 5.51% to 6.99%. Moreover, under optimal cultivation conditions, the recombinant IF2-Cas9 protein was produced in greater quantities than Cas9 alone (18.50 versus 13.65 mg/L). The solubility analysis revealed that SUMO-Cas9 exhibited higher solubility (9.25%) than Cas9 alone (5.51%), representing a 67.9 &plusmn; 8.2% increase in soluble expressio.</span></span></span><b> <span style="font-size:10.0pt"><span style="line-height:107%"><span new="" roman="" style="font-family:" times="">Conclusion:</span></span></span> </b><span style="font-size:10.0pt"><span style="line-height:107%"><span new="" roman="" style="font-family:" times="">While both fusion partners enhanced Cas9 expression in <i>E. coli</i>, SUMO demonstrated superior solubility-enhancing capability compared to IF2; however, the functional activity of the purified protein remains to be confirmed.</span></span></span></div> SpCas9 expression, CRISPR/Cas9, Initiation factor 2, Solubility-enhancing tag, Small ubiquitin-like modifier, Escherichia coli 149 158 http://jommid.pasteur.ac.ir/browse.php?a_code=A-10-632-4&slc_lang=en&sid=1 Farzaneh Barkhordari barkhordari@pasteur.ac.ir 0000-0003-0500-1726 No Biotechnology Research Center, Pasteur Institute of Iran, Tehran, Iran Mahshid Hosseini Shalmaei 0009-0002-9279-1294 No 1Biotechnology Research Center, Pasteur Institute of Iran, Tehran, Iran; 2Department of Biotechnology, Faculty of Advanced Science and Technology, Tehran Medical Sciences, Islamic Azad University, Tehran, Iran Setayesh Naderi No 1Biotechnology Research Center, Pasteur Institute of Iran, Tehran, Iran; 2Department of Biotechnology, Faculty of Advanced Science and Technology, Tehran Medical Sciences, Islamic Azad University, Tehran, Iran Mozhgan Raigani m_raigani@pasteur.ac.ir 0000-0003-4764-0606 No Biotechnology Research Center, Pasteur Institute of Iran, Tehran, Iran Yeganeh Kayalha 0009-0001-6196-9750 No Faculty of Life Sciences and Biotechnology, Shahid Beheshti University, Tehran, Iran Elham Bayat elhambyt@gmail.com No Biotechnology Research Center, Pasteur Institute of Iran, Tehran, Iran Negin Sadat Hashemi Aval n.hashemiaval@gmail.com 0009-0008-4727-8225 No Biotechnology Research Center, Pasteur Institute of Iran, Tehran, Iran Ahmad Adeli adelia@gmail.com 0009-0003-0213-6788 No Biotechnology Research Center, Pasteur Institute of Iran, Tehran, Iran Fatemeh Davami f.davami@gmail.com 0000-0002-7693-2926 Yes Biotechnology Research Center, Pasteur Institute of Iran, Tehran, Iran