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Volume 11, Issue 2 (Vol.11 No.2 Jul 2022)
rbmb.net 2022, 11(2): 310-319 |
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Zhansaya A, Malika N, Boris D, Kanat T, Kanatbek M, Yerlan R et al . Expression of Recombinant CTLA-4 and PD-L1 Proteins Fused with Thioredoxin, and Determination of Their Ligand-Binding Activities. rbmb.net 2022; 11 (2) :310-319
URL: http://rbmb.net/article-1-882-en.html
URL: http://rbmb.net/article-1-882-en.html
Adish Zhansaya * 
, Nurtleu Malika , Dzantiev Boris , Tursunov Kanat , Mukantayev Kanatbek , Ramankulov Yerlan , Mukanov Kasym
, Nurtleu Malika , Dzantiev Boris , Tursunov Kanat , Mukantayev Kanatbek , Ramankulov Yerlan , Mukanov Kasym
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.
Abstract: (2730 Views)
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.
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.
Type of Article: Original Article |
Subject:
Biochemistry
Received: 2022/02/17 | Accepted: 2022/02/23 | Published: 2022/08/7
Received: 2022/02/17 | Accepted: 2022/02/23 | Published: 2022/08/7
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