Authors:

Alex Author

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The interaction between programmed death ligand 1 (PD-L1) and programmed death 1 (PD-1) inhibits immune responses and promotes tumorigenesis. Therefore, this immune checkpoint has been considered a significant target for the treatment of cancers. Previously we have identified a specific canine immunoglobulin G (cIgG) antibody that targets the canine PDL1 (cPD-L1) and showed a significant effect on the inhibition of cancer cell proliferation. However, the precise cPD-L1 epitope and the interaction between the cIgG and cPD-L1 are unknown. In this study, we aim to determine the complex structure of the cIgG-PD-L1 using X-ray crystallography. The primary focus is to express the N-terminal domain of cPD-L1 (cPD-L1-ND) in the inclusion bodies using the E. coli expression system, followed by refolding procedures to obtain monomeric cPD-L1-ND proteins. This can be used to form a complex with the Fab fragments derived from the cIgG antibodies. A variety of refolding conditions were screened by several assays to attempt to purify and identify the properly refolded proteins. Currently, rapid dilution of the denatured proteins into pure water gives the best refolding result, yielding 2% monomeric cPD-L1-ND proteins. Alternatively, we have attempted to produce native-folded cPD-L1-ND proteins using the bac-to-bac insect cell expression system. We have obtained enough baculovirus stocks for large-scale protein expression. Additionally, commercially available full-length cPD-L1 was purchased and can be used for screening crystallization conditions of cPD-L1-Fab crystals. Successful structural determination will provide insight into the binding of cIgG and cPD-L1, which will benefit the development of immune checkpoint inhibitors.

Source:

Purdue University / 2023

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Co-authors:

Alex Author