Academic Papers
(AutoPre25D, RECEPTOR-NUT & KINASE-BAK1 Protein purificatio）Yifan Bai, et al. Structural basis for the activation of an LRR receptor by a single-disulfide-bridged peptide signal. Plant Communications. 2026.
2026-08-17 13:50
(AutoPre25D, RECEPTOR-NUT & KINASE-BAK1 Protein purificatio） Yifan Bai, et al. Structural basis for the activation of an LRR receptor by a single-disulfide-bridged peptide signal.
Plant Communications. 2026. https://doi.org/10.1016/j.xplc.2026.101932
Intramolecular disulfide bonds can modulate overall peptide rigidity and improve structural stability, thereby serving as essential post-translational modifications for signaling small secreted peptides (SSPs). Despite the widespread distribution of signaling SSPs with a single intramolecular disulfide bond, such as oxytocin, urotensin Ⅱ, vasopressin, and somatostatin in animals, few analogous SSPs have been characterized in plants (Daly and Wilson, 2021). To date, only delta-like phytocytokines (DEPs) (Wang et al., 2025) have been functionally characterized as such SSPs in plants. SMALL PHYTOCYTOKINES REGULATING DEFENSE AND WATER LOSS (SCREW) (Liu et al., 2022)/CTNIPs (Rhodes et al., 2022) are phytocytokines that activate immune responses (e.g., mitogen-activated protein kinase [MAPK] phosphorylation) and regulate stomatal closure through PLANT SCREW UNRESPONSIVE RECEPTOR (NUT)/HAESA-LIKE 3 (HSL3), which belongs to the leucine-rich repeat receptor-like kinase (LRR-RLK) XI subfamily. SCREW peptides activate NUT by promoting its heterodimerization with BRASSINOSTEROID INSENSITIVE 1-ASSOCIATED KINASE 1 (BAK1) (Liu et al., 2022). However, whether the biological function of SCREW2 depends on conformational changes induced by its disulfide bond and the underlying mechanisms governing the assembly of NUT–SCREW–BAK1 complexes remain elusive.....
The pFastBac1 vector was transformed into the DH10 Bac chemically competent cells (Coolaber Science & Technology). Recombinant bacmid were transfected into insect cells in the logarithmic growth phase, using linear polyethylenimine MW 40000 (40815ES08, Yeasen). Recombinant baculoviruses were added to high five cells in SIM HF Expression Medium (for Hi5, serum‑free; Sino Biological Inc.), containing 0.5% penicillin‑streptomycin (P1400, Beijing Solarbio Science & Technology Co., Ltd.). Liquid medium transfer was performed using a 25 mL serological pipette (H812004, BaiDi Biotechnology Co., Ltd.). Insect cells used for protein expression were cultured at 22 °C for 60 hours. Cell cultures were harvested and centrifuged at 4000 rpm for 20 min. The supernatants were purified using Ni‑NTA Beads (Smart‑Lifesciences, SA004050) at 4 °C. Target proteins were then eluted with 25 mM Tris (pH 8.0), 150 mM NaCl, and 250 mM imidazole. Proteins were further concentrated using ultrafiltration centrifuge tubes (Guangzhou Jet Bio‑Filtration Co., Ltd.). The eluates were purified by size‑exclusion chromatography using a HiLoad 16/60 Superdex 200 prep grade column (Cytiva, Unique AutoPre25D system, Inscinstech) equilibrated with 10 mM Bis‑Tris (pH 6.0) and 100 mM NaCl. Fractions corresponding to the target peaks were mixed with the 5X SDS‑PAGE Loading Buffer (Non‑Reducing, AIWB‑0025N, Swiss Affinibody LifeScience AG) and analyzed by SDS‑PAGE, followed by staining with Coomassie Brilliant Blue rapid staining solution (Lablead, G1042).
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