Directed evolution of Clec12a-binding peptides using mammalian-expressed recombinant protein

TL;DR

Researchers developed a method to produce a mouse protein called Clec12a in mammalian cells and used it to find peptides that bind to it, which could help in targeting immune cells.

Contribution

A scalable mammalian expression system for Clec12a was developed, enabling directed evolution of functional peptide ligands.

Findings

Murine Clec12a extracellular domain was produced at ~80 mg/L with proper glycosylation.

Three Clec12a-binding peptides were identified via ribosome display, with CBP2 showing highest affinity.

Selected peptides bound to primary dendritic cells, confirming native-like protein conformation.

Abstract

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 ∼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 (KD ≈ 6.29 μM). 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.

Image 1

•The murine Clec12a was expressed in mammalian cells in a yield of ∼80 mg/L.•The purified protein underwent N-glycosylation as a post-translational modification.•Recombinant Clec12a served as a native-like bait for ribosome display-based peptide screening.•Selected peptides bound to Clec12a-expressing primary dendritic cells.•The results indicated that the native folding of recombinant Clec12a was maintained.

The murine Clec12a was expressed in mammalian cells in a yield of ∼80 mg/L.

The purified protein underwent N-glycosylation as a post-translational modification.

Recombinant Clec12a served as a native-like bait for ribosome display-based peptide screening.

Selected peptides bound to Clec12a-expressing primary dendritic cells.

The results indicated that the native folding of recombinant Clec12a was maintained.

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