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Directed evolution of Clec12a-binding peptides using mammalian-expressed recombinant protein
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DOI：10.1016/j.bbrep.2026.102531.png)
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Abstract
En 中文
Clec12a is an immunoregulatory C-type lectin receptor expressed on dendritic cells as well as other myeloid cells, and involved in maintaining immune homeostasis. Unlike many other C-type lectin receptors that promote immune activation, Clec12a transmits inhibitory signals through an immunoreceptor tyrosine-based inhibitory motif, thereby contributing to immune regulation. Although the extracellular domain of murine Clec12a has been produced in previous studies, its suitability as a functional bait for directed evolution-based ligand discovery has not been established, nor have quantitative yield and scalability been reported. Here, we established a mammalian expression system that reproducibly yields similar to 80 mg/L of the mClec12a extracellular domain, providing material of sufficient quality and quantity for directed evolution of peptide binders. Incorporation of an interleukin-2 signal peptide enabled efficient secretion from Expi293 cells, and the purified protein retained N-glycosylation, indicating proper maturation through the endoplasmic reticulum-Golgi pathway. Using ribosome display with mClec12a as bait, three mClec12a binding peptides (CBP1-3) were isolated, with CBP2 showing the highest affinity (K-D approximate to 6.29 mu M). Importantly, CBP2 bound to primary dendritic cells derived from mouse spleen and bone marrow, supporting the native-like conformation and functional integrity of the recombinant mClec12a used for selection. The identified peptide represents a first-generation mClec12a(+) cell targeting ligand and provides a foundation for further optimization toward dendritic cell targeting and immunomodulatory applications.
Keywords:
Clec12a
Mammalian expression
Ribosome display
Directed evolution
Peptide binder screening
Journal
B
IF:
2.2
Papers:
291
Citations:
1