<scp>SNAC</scp> ‐Tag Integration Into <scp>NPN</scp> Platform Facilitates Efficient Purification of Recombinant Proteins From <i>Nicotiana benthamiana</i>

Plants have emerged as promising bioreactors to produce recombinant proteins, offering a safe, scalable, and cost-effective platform for vaccines, antibodies, and other biologics (Fischer and Buyel 2020). Significant progress has been made in optimising the expression and accumulation of various target proteins in plant cells (Sainsbury et al. 2009; Song et al. 2024). However, the downstream purification of plant-derived recombinant proteins remains a major bottleneck to industrial-scale production, which has been suggested in some cases to represent a major portion of the total production cost, occasionally approaching 80% (Buyel 2019), greatly limiting the commercial viability of plant-based systems. For plant-derived vaccines, such as trimeric hemagglutinin (HA, Song et al. 2021) and trimeric spike (S, Song et al. 2022) proteins, yields can reach 150–250 mg/kg of fresh plant tissue. Despite these promising expression levels, conventional Ni2+-NTA affinity chromatography often results in poor recovery (Song et al. 2022), which hinders the development and application of plant-derived vaccine candidates. In addition, cleaving affinity tags using proteases such as enterokinase, tobacco etch virus (TEV) protease, or thrombin protease after protein purification further increases the overall cost (Dang et al. 2019). To address these challenges, we incorporated the sequence-specific nickel-assisted cleavage (SNAC)-tag (Dang et al. 2019) into a natural peptidoglycan nanoparticle (NPN)–based purification platform (Song et al. 2025). This approach employs NPN to efficiently enrich target proteins from plant total protein extracts, followed by Ni2+-mediated chemical cleavage to cost-effectively and precisely remove the C-terminal NPN-binding domain, thereby releasing the target proteins (Figure 1A). Specifically, the NPN-SNAC platform achieves a fivefold higher recovery of tag-free GFP at around fiftieths of the cost compared with Ni2+-NTA purification. To test the NPN-SNAC platform, we first cloned the GFP gene into the pTEX1L vector containing the FD-SCpl7. This enabled the expression of GFP-FD-SCpl7, a recombinant protein that strongly binds NPNs. Additionally, we introduced a SNAC-tag (-GSHHW-) at the 3′ end of the FD domain to allow removal of the C-terminal SCpl7 domain, generating GFP-FD-SNAC-SCpl7 (Figure 1B). Both GFP-FD-SCpl7 and GFP-FD-SNAC-SCpl7 were transiently expressed in N. benthamiana, showing comparable expression levels (Figure 1C,D), which was approximately 1.2 g/kg of fresh weight (Figure S1). To evaluate the SNAC-tag cleavage efficiency, we incubated GFP-FD-SNAC-SCpl7 with the Ni2+-containing buffer over time. After 12 h, only 7% of the full-length protein remained (Figure 1E,F). We further optimised cleavage conditions by testing different temperatures (4°C, 22°C and 28°C). Cleavage was the most efficient at 28°C (Figure S2). Notably, SDS-PAGE revealed two distinct bands (F1 and F2) post-cleavage. Western blot analysis with anti-GFP antibody confirmed both fragments contained GFP (Figure 1G). To determine whether these bands resulted from non-specific cleavage, we performed protein C-terminal sequencing of F1 and F2 (Figure 1H,I). Both F1 and F2 were identified to contain peptide with the SNAC remaining residue, and their relative abundances were consistent (Figures S3 and S4). This indicates the molecular mass difference was not due to unintended C-terminal cutting. After Ni2+-mediated cleavage, we observed that a substantial portion of the cleaved GFP remained bound to the NPN through non-specific interactions, preventing its release. To address this, we tested various approaches, including adding different ionic or nonionic detergents (Triton X-100, Tween 20, SDS, CTAB) and adjusting the pH with HCl or Tris to alter protein charge after cleavage. We found that introducing 0.05% SDS (w/v), or supplementing with Tris (50–100 mM), resulted in an approximately twofold improvement in the efficiency of cleaved protein release from the NPN (Figure S5). To confirm the generalisability of the NPN-SNAC strategy, we replaced GFP with S and HA genes, creating S-FD-SCpl7, S-FD-SNAC-SCpl7, HA-FD-SCpl7 and HA-FD-SNAC-SCpl7 constructs (Figures S6A and S7A). Protein expression analysis showed comparable levels across all constructs, confirming that SNAC-tag insertion does not affect expression (Figures S6B,C and S7B,C). Unlike recombinant GFP (rGFP), both recombinant S and HA proteins displayed a single distinct band after cleavage, confirming the precision of the cleavage process. Due to the large size of the S protein, cleavage was difficult to distinguish and quantify (Figure S6D), so further functional validation focused on the HA protein. A time-course experiment showed that 90% of HA-FD-SNAC-SCpl7 was cleaved within 10 h (Figure S7D,E). Cleavage at 28°C for 10 h yielded approximately 76% of the cleaved protein (Figure S8). To benchmark the NPN-SNAC system against traditional methods, we compared its purification efficiency and cost-effectiveness to that of Ni2+-NTA affinity chromatography, a widely used method for purifying recombinant proteins. Total soluble proteins (TS) extracted from 0.05-g plants expressing GFP-FD-SNAC-SCpl7 or HA-FD-SNAC-SCpl7 were incubated with either NPNs (2 units, details in M&M) or Ni2+-NTA resin (50 μL) for 15 min (Figure 1J). Western blotting revealed that NPNs captured nearly all target proteins, whereas Ni2+-NTA left significant detectable unbound (Figure 1K and Figure S9A), even though 50 μL Ni2+-NTA resin is recommended to bind up to 2.5 mg of His-tagged protein from cleared cell lysates (Ni-NTA Agarose). Coomassie staining further confirmed that NPNs bound significantly more protein than Ni2+-NTA: 2.2 times more GFP-FD-SNAC-SCpl7 (Figure 1L,M) and 1.8 times more HA-FD-SNAC-SCpl7 (Figure S9B,C). For final field analysis, equal amounts of infiltrated plant tissues (0.05 g) expressing GFP-FD-SNAC-SCpl7 were subjected to purification using either the NPN-SNAC system (2-unit NPN) or conventional Ni2+-NTA (50 μL resin) chromatography (Figure 1N). The SDS-PAGE result showed that tag-free GFP obtained via NPN-SNAC was fivefold higher than the classical Ni2+-NTA approach (Figure 1O,P). Moreover, the NPN-SNAC method achieved these results at around fiftieths of the cost (Table S1). These results demonstrate that the NPN-SNAC platform exhibits less cost and higher purification yield than the conventional Ni2+-NTA system, while exhibiting minimal dependence on specialised equipment, thereby supporting its potential as a scalable approach for purifying recombinant proteins from plants. Notably, in this study, the cost comparison between the two purification methods was based on the reagents we used and calculated for a single purification run in the lab. Given the reusability of Ni2+-NTA, the actual cost comparison in scale-up applications in the factory should be evaluated with caution. Additionally, although the SDS-PAGE results showed a clear and single band after cleavage, it remains essential to perform appropriate functional validation for each target protein during the product development process. S.J.S. contributed to the conception of the study and wrote the manuscript. H.-X.M. and H.-P.D. contributed significantly to experiments and data analysis with the help of J.-H.S., S.-Y.R. and X.-J.X. Y.F.G. reviewed and revised the text. This research was supported by the Science and Technology Foundation of Beijing Life Science Academy (2024200CB0110), the Natural Science Foundation of Shandong Province (ZR2024QC068). This work was supported by the Natural Science Foundation of Shandong Province (ZR2024QC068) and Beijing Life Science Academy (2024200CB0110). The authors declare no conflicts of interest. The data that support the findings of this study are available on request from the corresponding author. The data are not publicly available due to privacy or ethical restrictions. Appendix S1: pbi70499-sup-0001-AppendixS1.docx. Please note: The publisher is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article.