GST Pull-Down, Co-Immunoprecipitation, Yeast Two-Hybrid, and Bimolecular Fluorescence Complementation: Investigating Protein Interactions and Subcellular Localization

Protocol 1: GST Pull-Down and Co-Immunoprecipitation

To investigate the interaction between SsCP1 and AtPR1, GST pull-down and co-immunoprecipitation assays were performed. Plasmids encoding GST-SsCP1 and His-AtPR1 were separately introduced into Escherichia coli strain BL21 (DE3), and protein expression was induced with 0.5 mM IPTG at 20ﾰC for 8 hours. Following sonication, equal amounts of lysates were mixed with high-affinity GST resin and incubated at 4ﾰC overnight with rotation. The bound proteins were then eluted with 10 mM glutathione elution buffer. Protein separation was achieved by 12% SDS-PAGE, followed by immunoblotting with anti-GST or anti-His antibody.

Protocol 2: Yeast Two-Hybrid System

To further confirm the interaction between SsCP1 and AtPR1, a yeast two-hybrid (Y2H) system was employed. SsCP1 (without signal peptide) was cloned into pGBKT7 as bait, and AtPR1 (without signal peptide) was cloned into pGADT7 as prey. These plasmids were co-transformed into yeast strain Y2H Gold. Truncated Sscp1 mutants with different deletions in the N-terminal region were also generated and subcloned into pGBKT7. Transformed yeast cells were assayed for growth on selective media and for the expression of reporter genes (X-a-gal and aureobasidin A). The presence of these reporter gene activities indicated interaction between the bait and prey proteins.

Protocol 3: Bimolecular Fluorescence Complementation and Subcellular Localization

A split-mCherry system was utilized for bimolecular fluorescence complementation (BiFC) analysis. N-terminal and C-terminal fragments of mCherry were subcloned into plasmids pCNF3 and pCNM3, respectively. Coding sequences of Sscp1 and AtPR1 were then introduced into these plasmids. BiFC was conducted by agroinfiltration in leaves of N. benthamiana, and protein expression was confirmed by Western blot.

For subcellular localization, coding sequences of Sscp1 and AtPR1 were introduced into pCNG and pCNR, respectively. Transient expression was achieved by agroinfiltration. Plasmolysis of N. benthamiana cells was performed using 1 M NaCl solution, followed by confocal microscopic analysis. Fluorescence in leaves was monitored at 3 days post-agroinfiltration using a confocal laser scanning microscope (CLSM). The excitation wavelengths used were 488 nm for eGFP and 587 nm for mCherry.