N. benthamiana plants were cultivated in soil under greenhouse conditions maintained at 23–24°C with 40–65% relative humidity and a 16-hour light/8-hour dark photoperiod. Leaves from 6- to 7-week-old plants were harvested for agro-infiltration experiments.

Actinidin is a vacuolar protein containing an N-terminal hydrophobic signal peptide required for cotranslational translocation into the ER lumen (Paul et al., 1995). In this study, we expressed the actinidin sequence (amino acids 25–381) without the N-terminal signal peptide, as this hydrophobic region could affect the solubility of the expressed protein. The GenBank accession number for the actinidin used in this study is MT463287. To generate the construct BiP-M-CBM3-bdSUMO-Actinidin-HA-HDEL (MCS-Actinidin), the sequence for Actinidin-HA-HDEL was synthesized (BIONICS Co., Ltd. Korea). A NaeI restriction site and a GGA codon for Gly were added at the 5′ end, while sequences coding for the HA tag, HDEL ER retention signal, TAA stop codon, and XhoI restriction site were added at the 3′ end. The synthesized actinidin-HA-HDEL was digested with NaeI and XhoI restriction endonucleases and ligated into a pUC-based vector pre-digested with the same enzymes. The pUC-based vector used in this study contained sequences coding for the BiP signal sequence, the M-domain of the human receptor-type tyrosine-protein phosphatase C, family 3 cellulose-binding module (CBM3), the small ubiquitin-like modifier (bdSUMO) cleavage site of Brachypodium distachyon, and the HSP transcriptional terminator (Nagaya et al., 2010; Islam et al., 2019). The resultant plasmid was further digested with XbaI and EcoRI and ligated into the pCambia 1300 binary vector, which had been digested with the same restriction enzymes. All restriction endonucleases were purchased from New England Biolabs (Ipswich, MA, USA).

The MCS-Actinidin construct was introduced into Agrobacterium tumefaciens (EHA105) via electroporation. The transformed A. tumefaciens cells were subsequently infiltrated into N. benthamiana leaves using syringe infiltration (Islam et al., 2019). During each agro-infiltration procedure, A. tumefaciens carrying the p38 gene, derived from the Turnip crinkle virus and encoding a suppressor of host gene silencing, was co-transformed to enhance expression.

The purification of the expressed actinidin was performed as previously described (Islam et al., 2019; Zaikova et al., 2022; Lee et al., 2024). Frozen leaf tissues (10 g fresh weight), harvested on days 3, 5 and 7 after agro-infiltration, were ground in liquid nitrogen. Total protein extracts were prepared by homogenizing the ground leaves in 30 mL of protein extraction buffer (50 mM Tris–HCl, pH 7.5, 150 mM NaCl, 1 mM DTT, 1% (v/v) Triton X-100, and 1× EDTA-free protease inhibitor cocktail (Roche, Switzerland)). After incubation at 4°C for 15 min, the extracts were filtered through Miracloth (Merck Millipore, USA). A 100 μL aliquot of the total extract was collected as the total (T) fraction. The remaining extracts were centrifuged at 19,400 ×g for 15 min at 4°C, and a 100 μL aliquot of the supernatant was collected as the soluble (S) fraction. The pellets were resuspended in 30 mL of protein extraction buffer, and a 100 μL aliquot was collected as the pellet (P) fraction. The soluble (S) fraction was used for purifying MCS-Actinidin with microcrystalline cellulose (MCC) beads (Sigma-Aldrich, St. Louis, MO, USA; CAS Number 9004-34-6) as described previously (Islam et al., 2019). Following MCC bead-based purification, the N-terminal domains containing CBM3 and bdSUMO were removed using His-bdSENP1 protease, which had been separately purified from E. coli BL21 (DE3) (Islam et al., 2019). All protein samples were analyzed by SDS-PAGE, followed by Coomassie Brilliant Blue (CBB) (BioShop, Canada; CAS Number 6104-59-2) staining or Western blotting using an anti-HA antibody (Roche, Basel, Switzerland; CAS Number 11867423001).

Western blotting was performed as described previously (Geem et al., 2021).

The purified actinidin was dialyzed against 0.1 M Tris-HCl, pH 7.0. Separately, 0.1 mg of β-casein (Merck & Co., Inc., USA) was dissolved in 0.1 M Tris-HCl, pH 7.0. Dialysis was carried out using Slide-A-Lyzer Dialysis Cassettes with a molecular weight cutoff of 10 kilodaltons (Thermo Scientific, catalog #66380). After dialysis, the concentration of actinidin was determined using a bicinchoninic acid assay kit (Abbkine, catalog no. KTD 3001, Wuhan, China). Subsequently, the yield of produced actinidin per biomass was calculated based on the initial mass of transformed leaves and the concentration of purified actinidin. Then, 7 μg of β-casein was incubated with 70 ng of the purified actinidin at the indicated temperatures and durations. The degradation of β-casein was analyzed by SDS-PAGE, followed by CBB staining. The band intensity of β-casein was quantified using ImageJ software (National Institutes of Health, USA).

In this study, we aimed to accumulate kiwifruit-derived actinidin in the ER lumen of N. benthamiana, a strategy that has proven successful for the expression of various foreign proteins in plants (Islam et al., 2019; Kim et al., 2022; Zaikova et al., 2022). To achieve ER localization, we incorporated the N-terminal signal peptide of binding immunoglobulin protein (BiP) and the C-terminal ER retention signal, HDEL, into the MCS-Actinidin construct ( Figure 1A ). Additionally, the M domain of the human receptor-type tyrosine-protein phosphatase C was included to enhance translational efficiency and stability of ER-localized proteins (Kang et al., 2018). For affinity purification, the CBM3 tag, which binds irreversibly to MCC beads, was added to the construct ( Figure 1A ) (Islam et al., 2019). Since the N-terminal region containing the BiP signal sequence, M domain, and CBM3 tag is unnecessary for actinidin activity post-purification, a bdSUMO domain was included before the actinidin-coding sequence to facilitate its removal. The bdSUMO domain enables specific cleavage by His:bdSENP1 protease, which recognizes the bdSUMO sequence and cleaves after the conserved two glycine residues (Islam et al., 2019). To ensure high expression levels, the expression cassette was driven by the cauliflower mosaic virus 35S promoter and terminated with the HSP transcriptional terminator ( Figure 1A ) (Nagaya et al., 2010).

The MCS-Actinidin construct was introduced into N. benthamiana leaves via agro-infiltration (Islam et al., 2019). At 3, 5, and 7 days post-infiltration, total protein extracts were prepared and subjected to centrifugation. The total, soluble, and pellet fractions were analyzed by Western blotting with an anti-HA antibody ( Figure 1B ). The majority of the expressed MCS-Actinidin was found in the soluble fraction, which facilitated the subsequent affinity purification process ( Figure 1B ). MCS-Actinidin was then purified from the soluble fraction using MCC beads ( Figure 1C ) (Islam et al., 2019). To remove the domains upstream of actinidin, the MCS-Actinidin conjugated to MCC beads was incubated with His:bdSENP1 protease, which recognizes the SUMO domain and cleaves immediately after Gly-Gly motif ( Figures 1A , 2A ) (Islam et al., 2019). After the cleavage reaction, the His:bdSENP1, which was no longer necessary, was removed using Ni²⁺-NTA beads ( Figure 2B ) (Islam et al., 2019). The yield of purified actinidin was approximately 10 mg per kg of fresh N. benthamiana leaves.

β-casein is a well-known substrate for kiwifruit-derived actinidin (Lo Piero et al., 2011; Puglisi et al., 2012). To verify the activity of actinidin expressed in N. benthamiana, β-casein was incubated with or without the purified enzyme at 37, 45, 55, and 65°C for 1 hour ( Figure 3A ). The purified actinidin exhibited the highest activity at 55°C ( Figure 3A ). Next, β-casein was incubated with or without the purified actinidin at 55°C for varying durations, ranging from 1 to 60 minutes ( Figure 3B ). Compared to the untreated control, β-casein was progressively degraded as the incubation time increased ( Figure 3B ).