Abstract
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.