en 34900145 <p style="text-align:justify"><span style="font-size:11pt"><span style="font-size:9.5pt">Background:</span> <span style="font-size:10.0pt">The recombinant human granulocyte colony stimulating factor conjugated with polyethylene glycol (PEGylated GCSF) has currently been used as an efficient drug for the treatment of neutropenia caused by chemotherapy due to its long circulating half-life. Previous studies showed that</span> <span style="font-size:10.0pt"><span style="background-color:white">Granulocyte Colony Stimulating Factor</span></span><span style="font-size:10.0pt"> (</span><span style="font-size:10.0pt">GCSF</span><span style="font-size:10.0pt">)</span><span style="font-size:10.0pt"> could be expressed as non-classical </span><span style="font-size:10.0pt">I</span><span style="font-size:10.0pt">nclusion </span><span style="font-size:10.0pt">B</span><span style="font-size:10.0pt">odies (ncIBs), which contained likely correctly folded GCSF inside at low temperature. Therefore, i</span><span style="font-size:10.0pt">n this study, a simple process was developed to produce PEGylated GCSF</span><span style="font-size:10.0pt"> from ncIBs.</span></span></p> <p style="text-align:justify"><span style="font-size:11pt"><span style="font-size:9.5pt">Methods:</span> <span style="font-size:10.0pt">BL21 (DE3)/pET-GCSF cells were cultured in the LiFlus GX 1.5 <em>L</em> bioreactor and</span><span style="font-size:10.0pt"> the expression of GCSF was induced by adding 0.5 <em>mM</em></span><span style="font-size:10.0pt"> IPTG. </span><span style="font-size:10.0pt">After 24 <em>hr</em> of fermentation, c</span><span style="font-size:10.0pt">ells were collected, resuspended, and disrupted. The insoluble fraction</span><span style="font-size:10.0pt"> was obtained from cell lysates and </span><span style="font-size:10.0pt">dissolved in 0.1% N-lauroylsarcosine solution</span><span style="font-size:10.0pt">. The presence and structure of dissolved GCSF were verified using SDS-PAGE, Native-PAGE, and RP-HPLC analyses. The dissolved GCSF was directly used </span><span style="font-size:10.0pt">for the conjugation with 5 <em>kDa</em> PEG<span style="background-color:white">. The PEGylated GCSF was purified using two purification</span></span> <span style="font-size:10.0pt"><span style="background-color:white">steps, including anion exchange chromatography and gel filtration chromatography.</span></span></span></p> <p style="text-align:justify"><span style="font-size:11pt"><span style="font-size:9.5pt">Results:</span> <span style="font-size:10.0pt"><span style="background-color:white">PEGylated GCSF was obtained with high purity (</span></span><span style="font-size:10.0pt"><span style="background-color:white">~</span></span><span style="font-size:10.0pt"><span style="background-color:white">97%) and was finally demonstrated as a form containing one GCSF molecule and one 5 <em>kDa</em> PEG molecule (monoPEG-GCSF).</span></span></span></p> <p style="text-align:justify"><span style="font-size:11pt"><span style="font-size:9.5pt">Conclusion:</span> <span style="font-size:10.0pt">These results clearly indicate that the process developed in this study might</span><span style="font-size:10.0pt"> be</span><span style="font-size:10.0pt"> a potential and practical approach to produce PEGylated GCSF from ncIBs expressed in </span><em><span style="font-size:10.0pt">Escherichia coli</span></em><em><span style="font-size:10.0pt"> (E. coli).</span></em></span></p> Granulocyte colony stimulating factor (G-CSF), Inclusion bodies, Polyethylene glycol 192 200 https://www.ajmb.org/En/Article.aspx?id=40477 https://www.ajmb.org/PDF/En/FullText/40477.pdf NguyenThi My TrinhVietnam National University, Ho Chi Minh City, Vietnam71800TranLinh ThuocVietnam National University, Ho Chi Minh City, Vietnam71801DangThi Phuong ThaoDepartment of Molecular and Environmental Biotechnology, University of Science, Ho Chi Minh City, Vietnam71802