Site is under construction

en Expression of Recombinant CTLA-4 and PD-L1 Proteins Fused with Thioredoxin, and Determination of Their Ligand-Binding Activities بیوشیمی Biochemistry مقالات اصلی Original Article <div style="text-align: justify;">Background: The use of chimeric proteins that selectively interact with various immune cell receptors to treat oncology patients has increased. One effective way to obtain recombinant proteins is to use the E. coli expression system. However, in eukaryotic protein production in E. coli, several difficulties arise that can be solved by fusing the target protein with thioredoxin. Thioredoxin can enhance solubility, but its large size can lead to an erroneous assessment of protein solubility, folding, and activity. The present study examined the ligand-binding activity of PD-L1, and CTLA-4 receptors fused with thioredoxin. Methods: The de novo synthesized genes of the extracellular domains of the PD-L1 and CTLA-4 were cloned into the pET28 and pET32 expression plasmids and used to transform E. coli BL21 cells. Purified recombinant proteins were characterized by western blotting, LC-MS/MS spectrometry, and ELISA. Results: Amino acid sequence comparisons of the recombinant proteins obtained by LC-MS/MS with the SwissProt database resulted in the highest comparison scores from 4950 to 13396. The binding efficiencies of recombinant human B7-1 Fc to rCTLA-4 and rTrx-CTLA-4 proteins in ELISA did not differ significantly. Similar results were obtained with recombinant rhesus monkey PD-1 hFc against rPD-L1 and rTrx-PD-L1. Conclusions: Recombinant proteins specifically reacted with recombinant human B7-1 Fc and recombinant rhesus monkey PD-1 hFc. The fusion of thioredoxin with recombinant proteins through linkers slightly affected the activity of the extracellular domains of CTLA-4 and PD-L1.</div> Chimeric Protein, CTLA-4, PD-L1, Protein Refolding, Recombinant Protein, Thioredoxin. 310 319 http://rbmb.net/browse.php?a_code=A-10-350-2&slc_lang=en&sid=1 Adish Zhansaya zhansaya.adish@gmail.com. 100319475328460012232 100319475328460012232 Yes National Center for Biotechnology, Kurgalzhyn road, 13/5, Nur-Sultan, 010000, Kazakhstan & L. N. Gumilyov Eurasian National University, Satpayev st., 2, Nur-Sultan, 010008, Kazakhstan. Nurtleu Malika 100319475328460012233 100319475328460012233 No National Center for Biotechnology, Kurgalzhyn road, 13/5, Nur-Sultan, 010000, Kazakhstan & L. N. Gumilyov Eurasian National University, Satpayev st., 2, Nur-Sultan, 010008, Kazakhstan. Dzantiev Boris 100319475328460012234 100319475328460012234 No A. N. Bach Institute of Biochemistry, Russian Academy of Sciences, Leninsky Prospect 33, Moscow, 119071, Russian Federation. Tursunov Kanat 100319475328460012235 100319475328460012235 No National Center for Biotechnology, Kurgalzhyn road, 13/5, Nur-Sultan, 010000, Kazakhstan. Mukantayev Kanatbek 100319475328460012236 100319475328460012236 No National Center for Biotechnology, Kurgalzhyn road, 13/5, Nur-Sultan, 010000, Kazakhstan. Ramankulov Yerlan 100319475328460012237 100319475328460012237 No National Center for Biotechnology, Kurgalzhyn road, 13/5, Nur-Sultan, 010000, Kazakhstan. Mukanov Kasym 100319475328460012238 100319475328460012238 No National Center for Biotechnology, Kurgalzhyn road, 13/5, Nur-Sultan, 010000, Kazakhstan.