Overview
Our V-CHARGEs system not only enables efficient ATP regeneration but also establishes a novel expandable multi-enzyme catalytic platform through P22-VLP encapsulation and external enzyme coupling.
We first assembled V-CHARGEs in vitro, then developed a simpler and more cost-effective in vivo assembly and separation method. Through rigorous testing, we confirmed the system's high efficiency and stability, and further validated its platform potential by coupling external enzymes.
Overall, V-CHARGEs serve as a typical representative of functional protein nanoparticles, applicable to high-value molecule production, artificial metabolic networks, and novel catalytic system development.
1.In Vitro Assembly
- We successfully expressed and purified SlPPK-SP and CP-SpyTag portein after extensive optimization.
- We explored the optimal conditions for in vitro assembly.
- We characterized the V-CHARGEs particles obtained from in vitro assembly.
Introduction and Objectives
This section aims to verify the feasibility of utilizing P22 Virus-Like Particles (P22-VLPs) for the in vitro assembly and encapsulation of SIPPK to construct in vitro assembled V-CHARGEs, and to characterize the assembly products structurally and functionally. We conducted work based on commonly used in vitro assembly protocols from the literature and performed extensive optimization and repeated experiments in three aspects: expression/purification, in vitro assembly, and characterization (SEC, DLS, TEM). Ultimately, we successfully obtained in vitro assembled V-CHARGEs particles with observable SIPPK encapsulation and verified their catalytic activity. However, we also identified and quantified bottlenecks in assembly efficiency and product homogeneity.
1.1 Plasmid Construction for SIPPK-SP and CP-SpyTag
We selected Escherichia coli (E. coli) BL21 (DE3) for the heterologous expression of SIPPK-SP and CP-SpyTag proteins. We constructed the following genetic pathways (Figure 1) and integrated the genes encoding the fusion proteins into plasmid backbones. We chose the pET-28a vector backbone. This T7 promoter-based vector contains a kanamycin resistance gene as a selectable marker and features an inducible lac operon (including the lacI repressor), ensuring that IPTG (IUPAC: Propan-2-yl-1-thio-β-D-galactopyranoside) can induce the expression of the fusion proteins. In the constructed plasmids, the C-terminus of the target gene (SIPPK-SP or CP-SpyTag) was fused with a His-tag to facilitate subsequent purification via nickel affinity chromatography. We used SnapGene software to design the plasmids (Figure 2),amplified the target gene fragments using primers synthesized by Sangon Biotech, and used In-Fusion Cloning technology to insert the gene fragments into linearized vectors.
Plasmids were extracted from E. coli DH5α and transformed into E. coli BL21(DE3) via heat shock. After transformation, the cells were inoculated on LB medium containing kanamycin for selection. After overnight incubation, we observed distinct bacterial colonies, indicating successful transformation. Finally, we identified the target genes by Polymerase Chain Reaction (PCR) and agarose gel electrophoresis. The gel electrophoresis image showed that the lengths of both SIPPK-SP and CP-SpyTag were between 1000 bp and 2000 bp (Figure 3), consistent with their actual sizes.The successfully transformed plasmids were sent to Sangon Biotech for sequencing, and the sequencing results confirmed that the target gene fragments were successfully inserted into the plasmids.
Summary
We successfully constructed the recombinant plasmids pET-28a::SlPPK-SP-FLAG-His and pET-28a::CP-SpyTag-His for expressing SIPPK-SP and CP-SpyTag, and transformed them into the expression strain BL21(DE3), laying the foundation for the subsequent heterologous expression and purification of the two proteins.
1.2 Expression and Purification of SIPPK-SP and CP-SpyTag
The heterologous expression of SIPPK-SP and CP-SpyTag proteins was particularly challenging compared to other proteins, as we faced significant issues with inclusion body formation. The expression of SIPPK-SP was highly prone to forming inclusion bodies, which is closely related to SIPPK's natural tetrameric state, large molecular weight (monomer ~50 kDa, tetramer ~200 kDa), and its folding/oligomerization requirements. After attempting inclusion body refolding, optimizing induction conditions, and improving protein purification procedures (Engineering Wet Lab1), we successfully developed a set of induction conditions and a mature protein purification method to purify both proteins.
The overnight-cultured seed culture was inoculated into fresh LB medium at a ratio of 1:100, and cultured at 37 °C with shaking at 200 rpm until the OD₆₀₀ reached 0.6–0.8. Then, 0.1 mM isopropyl-β-D-thiogalactopyranoside (IPTG) was added to induce protein expression, and cultivation continued at 16°C, 200 rpm for 16 h. Bacterial cells were collected by centrifugation at 6000 rpm for 10 min, resuspended in Buffer A (50 mM Tris-HCl, pH 8.0, 300 mM NaCl, 3 mM β-mercaptoethanol), and subjected to ultrasonic disruption (cycle of 3 s sonication / 6 s interval, total 10 min). The lysate was centrifuged at 12000 rpm for 45 min, and the supernatant and pellet were collected separately for subsequent purification.
We used nickel column affinity chromatography to isolate and purify the proteins from the supernatant. First, proteins were eluted stepwise using imidazole gradients ranging from 100 mM to 500 mM. Then, SDS-polyacrylamide gel electrophoresis (SDS-PAGE) was performed on the protein solution, flow-through, and eluates from different imidazole concentrations to analyze protein expression. To quantify the concentration of the extracted proteins, we used the Bradford method to determine the concentration of the purified proteins.
From the SDS-PAGE results of SIPPK-SP and CP-SpyTag (Figure 4), it can be observed that 500 mM imidazole eluted the SIPPK-SP protein from the column, and 300 mM imidazole eluted the CP-SpyTag protein from the column. At these concentrations, the purified proteins were relatively pure with minimal contaminating proteins. And we superisely observe that the molecular weight of SIPPK-SP and CP-SpyTag is too close to seperate, which makes a little trouble in later experiments.
We also characterized the particle size of the CP-SpyTag self-assembly products by Dynamic Light Scattering (DLS) and characterized their shape and particle size using Transmission Electron Microscopy (TEM) (Figure 5). We found that most CP-SpyTag could self-assemble into VLPs with particle sizes ranging from 50 to 500 nm. This demonstrates that we successfully expressed and purified CP-SpyTag, and also proves that CP-SpyTag can self-assemble into spherical structures in vitro, increasing the feasibility of subsequently encapsulating SIPPK-SP using VLPs.
Summary
By optimizing expression conditions and purification methods, we successfully obtained highly pure, soluble SIPPK-SP and CP-SpyTag proteins from E. coli, providing the key materials for the in vitro assembly of SIPPK-SP and CP-SpyTag. We also successfully characterized the self-assembly products of CP-SpyTag, strengthening our confidence in achieving VLP encapsulation of enzymes.
1.3 Assembly and Characterization
After obtaining the two proteins, we performed in vitro assembly of SlPPK-SP and CP-SpyTag. Equal volumes of guanidine hydrochloride and CP-SpyTag protein solution were mixed and incubated on ice for 30 minutes. Then, SlPPK-SP was added at a specific ratio and mixed thoroughly. Considering that the SP:CP ratio varies in different literatures, we tested different ratios and finally determined that a concentration ratio of 1:2 yielded the best assembly results, producing in vitro assembled V-CHARGE particles. Two rounds of dialysis were performed to remove most of the unassembled protein. The specific assembly principle is shown in Figure 6. To further improve the purity of the in vitro assembled V-CHARGEs, we used size exclusion chromatography to purify the assembly mixture and analyzed the obtained, higher-purity V-CHARGEs by SDS-PAGE (Figure 7).
We characterized the particle size of the in vitro assembled V-CHARGEs by Dynamic Light Scattering (DLS) and used Transmission Electron Microscopy (TEM) to characterize the assembly status, including shape and particle size (Figure 8), comparing these results with the characterization results of VLPs assembled solely from CP.
DLS and TEM analysis results showed that the particle size of the in vitro assembled V-CHARGEs was around 200 nm. However, there were many small particles (size < 10 nm) present in the system, likely indicating that a significant amount of SIPPK-SP was not encapsulated into the spheres assembled by CP-SpyTag but remained dispersed in the system, suggesting suboptimal in vitro assembly efficiency.
To achieve better assembly results, we further explored the assembly conditions in detail (Engineering Wet Lab2). However, we found that regardless of optimization efforts, we could not achieve efficient assembly to obtain large quantities of in vitro assembled V-CHARGEs particles.
Summary
Using in vitro co-assembly (mixing SIPPK-SP and CP-SpyTag at different ratios, incubating under appropriate buffer and temperature conditions), we were able to observe the target in vitro assembled V-CHARGEs peak and collect the corresponding fractions via SEC. Transmission Electron Microscopy (TEM) showed recognizable hollow or solid nanoparticles, and density (dark regions) could be detected inside some particles, inferred to be successful encapsulation of SIPPK. However, the assembly products exhibited high polydispersity: a large number of small particles coexisted with unassembled proteins, resulting in a relatively overall low assembly yield.
1.4 Discussion
Summary and Reflection
In this part of the study, we successfully constructed and expressed the SIPPK-SP and CP-SpyTag fusion proteins. By optimizing induction conditions and purification processes, we effectively addressed the severe inclusion body problem, establishing a unique expression and purification strategy suitable for large-molecular-weight proteins. Through nickel affinity chromatography, size exclusion chromatography and other steps, we obtained target proteins with relatively high purity and completed the in vitro assembly and characterization of V-CHARGEs and their characterization. Dynamic Light Scattering and Transmission Electron Microscopy results demonstrated that SIPPK-SP could be successfully encapsulated within P22-VLPs. This is the first time that a tetrameric enzyme with a molecular weight of up to 200 kDa has been encapsulated in the P22-VLP system, providing experimental evidence for expanding the application scope of this system.
However, we also identified a clear issue of insufficient assembly efficiency: despite exploring different assembly ratios and conditions, the system still contained numerous small particles or incompletely assembled structures, indicating significant steric hindrance limitations for large, multimeric proteins during the in vitro assembly process. This result contrasts sharply with the encapsulation efficiency reported in the literature for small to medium-sized monomeric enzymes like GFP or alcohol dehydrogenase, suggesting that we need to overcome the limitations of traditional in vitro assembly strategies when constructing more complex enzyme systems.
Possible Mechanisms Underlying Low Yield and Polydispersity
Causes of Low Yield and Polydispersity
1. Steric Hindrance: SIPPK has a high molecular weight and functions as a tetramer. The volume of a single SIPPK-SP and the space occupied by the formed tetramer might exceed the capacity of the P22 typical internal cavity under conditions without additional expansion. This could prevent some SP-SIPPK from being correctly incorporated or lead to misassembly. During normal P22 assembly, approximately 420 CP subunits and about 100–300 SP subunits cooperatively form a capsid with a diameter of about 58 nm; when the cargo volume approaches the cavity limit or the cargo is a rigid tetramer, the assembly pathway and product distribution can be significantly altered (naturally tending to produce unassembled proteins or aberrant particles). [1]
2.Multimer Kinetics and Folding: If SIPPK pre-forms tetramers or misfolds in vitro, the localization signal at the SP end might be masked, thereby reducing loading efficiency.
3.Kinetics and Concentration Effects: Assembly is a nucleation-and-growth process. The relative concentrations of CP and SP, as well as the total protein concentration, affect the nucleation rate and competition with correct assembly pathways. Excessively high protein concentrations might lead to "multiple nucleation" or condensate formation (which has been demonstrated to occur in studies of other VLP assemblies). [2]
Comparison with Literature
Previous studies have successfully encapsulated enzyme cascade systems inside P22 VLPs and demonstrated protection of enzyme activity and proximity effects. However, these successes mostly involved monomeric or smaller enzymes (or mild multimers); encapsulating large tetramers with volumes approaching or exceeding the internal cavity capacity presents new challenges. The pore and molecular diffusion characteristics of P22 also determine the entry and exit of small molecules and the permeation behavior of inhibitors (like Pi), which significantly impact the subsequent ATP/ADP cycle (the pore and molecular sieve effects of P22 have been quantitatively studied in the literature). [3]
Outlook
This phenomenon suggests that traditional in vitro assembly methods are inadequate for the efficient loading of large, multimeric enzymes. To break through this bottleneck, we plan to shift to an in vivo assembly strategy. Leveraging the natural folding and assembly processes within the cellular environment, we expect to achieve higher loading efficiency, more uniform particle distribution, and, based on this, conduct more in-depth enzyme activity studies.
2. In Vivo Assembly
- We co-expressed SP and CP proteins in the same E. coli strain to produce V-CHARGEs particles.
- We tested various purification methods and established a simple, low-cost production process.
- We characterized the V-CHARGEs particles obtained from in vivo assembly.
Introduction and Objectives
At the early stage of our project, we attempted to assemble V-CHARGEs particles in vitro. However, during the experiments, we encountered several challenges. On the one hand, protein extraction and purification inevitably caused significant loss, leading to low overall yield. On the other hand, the assembly efficiency was unsatisfactory: results from DLS and TEM revealed that most particles were incomplete with heterogeneous size distribution, which resulted in insufficient enzymatic activity. These difficulties are consistent with previous reports on the challenges of in vitro assembly of P22-VLPs, suggesting that this approach has inherent limitations when dealing with multi-enzyme complexes.
Based on these findings, we proposed an in vivo assembly strategy to address the above issues. Specifically, we co-expressed SlPPK-SP and CP-SpyTag in E. coli BL21(DE3), allowing them to spontaneously assemble into V-CHARGEs particles within the cell. Compared with in vitro assembly, in vivo assembly offers several potential advantages:
1. Higher assembly efficiency — Proteins can interact as they are synthesized, reducing the accumulation of unassembled monomers.
2. Structures closer to the native state — Folding and spatial arrangement in the cellular environment may help maintain enzymatic activity and stability.
3. Simplified workflow — Avoiding repeated in vitro purification and assembly steps, target particles can be obtained directly through affinity chromatography or ultracentrifugation.
4. Greater scalability — This strategy may provide a foundation for large-scale production and future industrial applications.
In subsequent experiments, we constructed both dual-plasmid and single-plasmid expression systems, and optimized induction conditions (stepwise induction with arabinose and IPTG). By combining Ni-affinity chromatography, sucrose density gradient ultracentrifugation, and size-exclusion purification, we successfully obtained relatively pure in vivo assembled V-CHARGEs particles from E. coli lysates. Further characterization by DLS and TEM confirmed particle formation, demonstrating the feasibility and advantages of the in vivo assembly strategy.
Conception of the Idea
During our experiments, we found that in vitro assembly of V-CHARGEs faced several obstacles. First, protein purification inevitably led to protein loss and contamination by non-specific proteins; even after repeated purification with size-exclusion and Ni-affinity chromatography, it was difficult to obtain highly pure assembly products. Second, the assembly efficiency was low: DLS and TEM revealed mostly incomplete or size-heterogeneous substructures, resulting in limited enzymatic activity. Similar issues have been reported in studies of in vitro assembly of P22-VLPs [4,5], suggesting that in vitro assembly inherently struggles with large macromolecular complexes or multi-enzyme systems.
To overcome these challenges, we explored an alternative strategy: co-expressing SlPPK-SP and CP-SpyTag in E. coli BL21(DE3), enabling spontaneous in vivo assembly of V-CHARGEs particles. This approach avoids protein loss and inefficiency associated with in vitro assembly, achieving “on-site” assembly of the particles. We then aimed to isolate and enrich in vivo assembled V-CHARGEs directly from cell lysates via Ni-affinity chromatography or sucrose gradient ultracentrifugation.
Literature review revealed that in vivo assembly has been previously employed in constructing P22-VLP complexes, with reported advantages. For example, P22 coat protein can interact with scaffold protein and assemble directly within E. coli cells [6]; similarly, certain engineered fusion proteins can achieve spontaneous encapsulation upon co-expression [7]. These findings indicate that in vivo assembly, by allowing proteins to interact immediately upon synthesis in the same environment, significantly improves assembly efficiency and reduces unassembled intermediates. Moreover, the resulting particles are more likely to adopt native-like folding and conformations, potentially enhancing enzymatic stability and activity. Most importantly, this method eliminates multiple purification and assembly steps, simplifying the workflow and offering better scalability and industrial potential.
In summary, we hypothesize that in vivo assembly not only overcomes the low yield and purification difficulties of in vitro assembly, but may also offer unique advantages in maintaining enzymatic activity and enabling scalable production. In future work, we will continue optimizing the in vivo co-expression system and validate its performance through systematic biochemical and structural characterization.
2.1 In Vivo Assembly Plasmid Construction
We constructed the following genetic pathway (Figure 9) and integrated the genes encoding fusion proteins into the plasmid backbone. In order to sequentially induce the expression of SlPPK-SP and CP-SpyTag carried by the dual-plasmid system in the bacterial cells, we additionally constructed an arabinose-inducible plasmid containing SlPPK-SP (Figure 10A). We chose the pBAD33 vector backbone, which is based on the arabinose promoter and contains a chloramphenicol resistance gene as a selection marker. It is also equipped with an arabinose operon to ensure that arabinose (IUPAC: L-(+)-Arabinose) can induce the expression of the fusion protein. For CP-SpyTag, we still used the pET-28a::CP-Spytag-His plasmid from the in vitro assembly, induced its expression by adding IPTG, and used kanamycin for selection.
At the same time, we also attempted to introduce SlPPK-SP and CP-SpyTag into a single plasmid (Figure 10B). We chose the pACYCDuet-1 vector backbone, which is based on the T7 promoter and contains a chloramphenicol resistance gene as a selection marker. It is also equipped with an inducible lac operon (including the lacI repressor) to ensure that IPTG can induce the expression of the fusion protein. However, due to time constraints, at the time of Wiki submission, we only successfully constructed the plasmid but were unable to carry out protein induction expression and related experiments, which will be continued later.
We used SnapGene software to design plasmids (Figure 10), amplified target gene fragments with primers synthesized by Sangon Biotech, and inserted gene fragments into linearized vectors using seamless cloning technology.
We prepared competent E. coli BL21(DE3) containing CP-SpyTag-His and transformed the obtained pBAD33::SlPPK-SP-FLAG plasmid into the prepared competent cells using the heat shock method. After transformation, the strains were inoculated onto LB agar plates containing chloramphenicol and kanamycin for selection. Since the plasmid carries the chloramphenicol resistance gene and the competent cells contain the kanamycin resistance gene, only those E. coli cells that successfully acquired the plasmid could grow on these plates. After overnight incubation, we observed distinct bacterial colonies, indicating successful transformation. Finally, we identified the target gene by Polymerase Chain Reaction (PCR) and agarose gel electrophoresis. The gel electrophoresis results showed that the lengths of SlPPK-SP and CP-SpyTag were both between 1000 bp and 2000 bp (Figure 11), consistent with their actual sizes. We submitted the successfully transformed plasmids to Sangon Biotech for sequencing, and the results confirmed that the target gene fragments were successfully and correctly inserted.
Summary
We successfully constructed dual-plasmid and single-plasmid systems expressing the two proteins SlPPK-SP and CP-SpyTag. Through resistance screening and PCR verification, we ensured the correct insertion of the genes, laying the foundation for the subsequent expression of these two proteins and the purification of products after in vivo assembly.
2.2 In Vivo Expression and Purification
In this part, we first adopted the method of sequential induction with arabinose + IPTG to induce the expression of SP-SlPPK and CP-SpyTag proteins. By exploring the induction expression conditions (including induction time, induction temperature, IPTG concentration added during induction (Engineering Wet Lab2), we obtained good expression results. Subsequently, we used Ni-bead affinity chromatography purification, ultracentrifugation purification, and sucrose gradient) ultracentrifugation to directly isolate and enrich in vivo assembled V-CHARGEs from cell lysates. Then, we characterized in vivo assembled V-CHARGEs particles using SEM, DLS, and TEM.
For the double-plasmid system, the overnight seed culture was inoculated into fresh LB medium at a ratio of 1:100, and incubated at 37 ℃, 200 rpm until OD600 reached 0.3–0.5. Subsequently, 0.2% arabinose was added to induce SlPPK-SP expression, and incubation was continued at 28 ℃, 200 rpm until OD600 reached 1.2–1.5. Then, 0.1 mM isopropyl-β-D-thiogalactopyranoside (IPTG) was added to induce CP-SpyTag expression, and incubation was continued at 16 ℃, 200 rpm for 12 h.
We hypothesize that under this sequential induction condition, SP-SlPPK proteins are first fully expressed in the cytoplasm, providing anchors and templates for the subsequent CP-SpyTag self-assembly. Once CP-SpyTag is induced, it gradually assembles with SP-SlPPK inside the cell to form complete V-CHARGEs particles (Figure 12).
To confirm the successful induction expression and self-assembly of V-CHARGEs within host cells, the cell samples of the double-plasmid system after induction expression were sent to ServiceBio for ultrathin sectioning and transmission electron microscopy (TEM) detection. TEM results showed that a large number of uniform spherical particles with a diameter of about 50 nm could be observed in the cytoplasmic region of cell sections (Figure 13), whose morphology was highly consistent with the in vivo assembled V-CHARGEs nanoparticles subsequently obtained by purification. This result indicated that after sequential induction by arabinose and IPTG, SlPPK-SP and CP-SpyTag could effectively complete assembly in cells to directly form the target nanoparticle structure. This result provided intuitive evidence for the success of the in vivo assembly strategy.
After induction expression, the cells were collected by centrifugation at 6000 rpm for 10 min, resuspended in Buffer A (50 mM Tris-HCl, pH 8.0, 300 mM NaCl, 3 mM β-mercaptoethanol), and disrupted by ultrasonication (3 s on/6 s off cycle, for a total of 10 min). The lysate was centrifuged at 12000 rpm for 45 min, and the supernatant and precipitate were separately collected for subsequent purification.
We first used Ni-column affinity chromatography to separate and purify the proteins in the supernatant. First, proteins were eluted with 300 mM imidazole, and then the eluted fraction was analyzed by SDS-polyacrylamide gel electrophoresis (SDS-PAGE). The protein expression status was analyzed through gel electrophoresis results. Meanwhile, Western blotting was also performed to verify the correctness of the in vivo assembly product structure. Size-exclusion chromatography was further used to purify the protein, and the results are shown in Figure 14. Combined with WB results, we found that a large amount of SlPPK-SP was still present in the eluted fraction obtained after Ni-column affinity purification, while size-exclusion chromatography could effectively remove free SlPPK-SP.
To further verify whether in vivo assembled V-CHARGEs differ from in vitro assembled ones and empty shell CP, we performed native agarose gel electrophoresis of different protein samples under optimized conditions (upper 0.5% separating gel, lower 1% supporting gel, 8–10 V voltage, 10–15 h electrophoresis, run on ice). Clear protein migration bands were successfully obtained.
The results showed that the migration rates of in vivo assembled particles, in vitro assembled particles, and empty shell CP were different: in vitro assembled particles migrated slower than empty shell CP, while in vivo assembled particles migrated faster than empty shell CP (Figure 15). This difference indicated that SP-SlPPK successfully assembled with CP both in vivo and in vitro, but the assembly modes might be different.
To facilitate the extraction of in vivo assembled V-CHARGEs in industrial production, we referred to published methods of ultracentrifugation separation of Virus-Like Particles (VLPs) (Vicente et al., 2011; Peyret, 2015; Bakhshizadeh Gashti et al., 2024), and optimized them accordingly. Specifically, the induced cells were collected by centrifugation, disrupted by ultrasonication in Buffer A, centrifuged again to remove debris, and the obtained supernatant was subjected to sucrose density gradient ultracentrifugation (gradient from top to bottom: 20%, 35%, 45%, 50%, 60%, 5 mL each layer; 150,000 × g, 4 ℃, 6 h), as shown in Figure 16.
After ultracentrifugation, we obtained an obvious protein band in the 45% sucrose layer. The actual photograph and schematic diagram of the protein band are shown in Figure 17. We aspirated and retained the protein supernatant from the top layer after ultracentrifugation as sample No. 0, and then separated the remaining sucrose cushion into samples by aspirating 1 mL from top to bottom sequentially, numbered 1–26. All the obtained samples were subjected to SDS-PAGE to investigate the separation effect of ultracentrifugation. Based on the gel electrophoresis results, we also used ImageJ to quantitatively analyze the relative protein concentrations of the protein supernatants in different layers after ultracentrifugation.
According to the SDS-PAGE results (Figure 18), we obtained strong CP and SP-SlPPK bands at the position corresponding to the protein band (No. 16), with very few impurities. In the surrounding samples, CP and SP-SlPPK bands gradually weakened; at the bottom of the sucrose cushion (No. 26), we obtained very strong CP and SP-SlPPK bands, along with some impurities.
To confirm which of the different protein samples obtained after ultracentrifugation represented the best assembled product with complete assembly and almost no free SP-SlPPK, we used Dot Blotting for verification. The specific operation and principle are shown in Figure 19. Water (blank control), CP empty shell, purified SP-SlPPK, ultracentrifugation sample No. 16, and sample No. 26 were diluted to the same concentration (1 ng/μL), and 1 μL and 3 μL were spotted onto the PVDF membrane as replicates.
The detection results showed that the SP-SlPPK control exhibited a strong signal, confirming the validity of the detection system; CP and the blank control showed no signal. Comparing samples 16 and 26, sample 16 showed a very weak signal, while sample 26 showed a stronger signal (Figure 20), indicating that sample 16 contained little free or exposed SP-SlPPK, with higher particle assembly integrity; while sample 26 still contained a certain amount of exposed SP-SlPPK. Based on this result, we selected sample 16 for subsequent size-exclusion chromatography purification and further functional verification.
To further compare the enzymatic properties of different fractions obtained from ultracentrifugation, we measured the specific enzyme activities of samples 16 and 26 (Figure 21). The results showed that although sample 26 had a higher protein concentration, its specific enzyme activity (enzyme activity per unit protein content) was significantly lower than that of sample 16; conversely, sample 16 showed higher specific enzyme activity, indicating that the conformation of SlPPK within its particles was more intact and its function better preserved.
Combining the results of SDS-PAGE (Figure 18), Dot Blotting (Figure 20), and specific enzyme activity measurement (Figure 21), it can be seen that: in sample 16, CP and SP-SlPPK bands were clear and with few impurities; the Dot Blotting signal was extremely weak, indicating almost no free or exposed SP-SlPPK. In terms of assembly integrity and enzymatic performance, sample 16 was superior to sample 26. Therefore, we selected sample 16 as the representative assembled product of in vivo assembled V-CHARGEs for subsequent size-exclusion chromatography purification and functional verification experiments.
We further purified the in vivo assembled V-CHARGEs obtained after ultracentrifugation using size-exclusion chromatography. Based on the SDS-PAGE, Dot Blotting, and preliminary enzyme activity measurement results, we selected the protein band sample at the protein ring (sample No. 16), which had high target protein content, very few impurities, high particle assembly integrity, and high enzymatic activity, for further size-exclusion chromatography purification. The size-exclusion chromatography results are shown in Figure 22. The results indicated that the sample obtained at the protein band after ultracentrifugation eluted outside the column volume of the size-exclusion chromatography column, suggesting that it was successfully in vivo assembled V-CHARGEs. Furthermore, the sample showed very high purity with almost no impurity peaks.
Summary
In this part, V-CHARGEs were efficiently expressed and assembled in vivo using the double-plasmid system. Through TEM observation of ultrathin sections of optimized induced samples, a large number of uniform spherical particles with a diameter of about 50 nm were clearly captured in the cytoplasmic region of host cells, whose morphology was highly consistent with purified in vivo assembled V-CHARGEs.
Through preliminary Ni-column affinity chromatography and size-exclusion chromatography purification, we successfully obtained correctly in vivo assembled V-CHARGEs particles. We further confirmed through native gel electrophoresis that the migration rate of in vivo assembled products differed from in vitro assembled ones and CP empty shells, suggesting possible differences in assembly mode. By ultracentrifugation combined with sucrose density gradient separation, we obtained a large amount of in vivo assembled V-CHARGEs in a convenient manner. The protein band at fraction 16 corresponded to high purity, nearly no free SP-SlPPK, and significantly higher specific activity than the sediment fraction 26, indicating better assembly integrity and functional preservation. Finally, after further purification by size-exclusion chromatography, fraction 16 exhibited a single and highly pure peak, confirming it as successfully assembled and high-purity in vivo assembled V-CHARGEs. Overall, we established a stable and effective preparation process of V-CHARGEs from in vivo expression to purification, obtaining functional and high-purity representative assembled products, laying a solid foundation for subsequent functional studies.
2.3 Characterization
We characterized the particle size of in vivo assembled V-CHARGEs before and after ultracentrifugation by Dynamic Light Scattering (DLS), and characterized the assembly of in vivo assembled proteins including shape and particle size by Transmission Electron Microscope (TEM). DLS results showed that the particle size of in vivo assembled V-CHARGEs was about 150 nm. TEM images clearly showed approximately spherical nanoparticles with diameters similar to VLPs without encapsulated SlPPK. At the same time, compared with before ultracentrifugation, the particle size range of the samples after ultracentrifugation was smaller and the homogeneity was better, indicating that the in vivo assembled samples obtained after ultracentrifugation were relatively pure and the assembly effect was good.
Summary
DLS and TEM characterization confirmed the formation of in vivo assembled V-CHARGEs particles, with uniform particle size and complete structure, and better effect after ultracentrifugation. These results are consistent with expectations, indicating that the in vivo assembly strategy successfully produced functional nanoparticles, providing high-quality core materials for subsequent experiments to detect the enzymatic activity of SlPPK-SP encapsulated in in vivo assembled V-CHARGEs particles.
2.4 Discussion
Summary and Reflection
In this study, we successfully achieved the in vivo assembly of V-CHARGEs particles and established a complete purification and identification process. Compared with in vitro assembly, in vivo assembly shows the following significant advantages:
1. Higher assembly efficiency: SlPPK-SP and CP-SpyTag were co-expressed in cells and spontaneously assembled, reducing the accumulation of unassembled monomers;
2. Higher purity and stability: Through the combined application of affinity chromatography, sucrose density gradient ultracentrifugation, and size-exclusion chromatography, we were able to stably obtain high-purity particles with significantly reduced contaminating proteins;
3. Closer to natural conformation: DLS and TEM results showed that in vivo assembled particles had uniform particle size and complete spherical structures, indicating that this strategy could maintain correct folding and spatial conformation in the cellular environment.These results verified the feasibility of in vivo assembly and also indicated its greater potential compared with in vitro assembly when dealing with complex multi-enzyme systems. Meanwhile, the process of combining ultracentrifugation and size-exclusion chromatography provides a technical basis for subsequent large-scale applications.
Outlook
In vivo assembly not only provided us with high-purity and complete V-CHARGEs particles, but more importantly, laid a reliable foundation for functional detection. Our next research will focus on the enzymatic properties of SlPPK-SP inside the particles. Through enzymatic property experiments, we will verify the enhancement effect of CP shell encapsulation of SlPPK on its activity and stability, and further reveal the application potential of V-CHARGEs as a multi-enzyme complex platform.
3. Enzyme Activity Validation
- We verified that V-CHARGEs can tolerate acidic environments, high phosphate concentrations, and protease degradation.
Introduction and Objectives
In the in vivo assembly section, we successfully isolated and purified high-purity, intact in vivo assembled V-CHARGEs particles, laying a reliable foundation for functional assays. In this part, we systematically compared the ATP production capacity of free SlPPK and in vivo assembled V-CHARGEs under prolonged reaction times and various stress conditions (pH, Pi, protease).
This section utilized a firefly luciferase-based ATP detection kit to measure ATP levels under different experimental conditions, thereby reflecting the activity of the relevant enzymes. First, a standard curve correlating ATP concentration with luminescence intensity was established to ensure the accuracy and reliability of ATP quantification. Based on this, the activities of free SlPPK and in vivo assembled V-CHARGEs were compared, and the effects of different protease concentrations, pH, and phosphate levels on ATP generation were further investigated.
The results indicated that SlPPK encapsulated within in vivo assembled V-CHARGEs exhibited significantly higher ATP levels compared to free SlPPK under high phosphate concentrations and protease treatment, demonstrating their protective effect on the enzyme. Furthermore, under acidic conditions (pH 3.0~5.5), the ATP levels of in vivo assembled V-CHARGEs were also superior to those of free SlPPK, highlighting their stability advantage against acid stress. Concurrently, control experiments showed that firefly luciferase activity was largely unaffected by different pH, phosphate, and protease conditions, further confirming the reliability of the experimental results.
3.1 ATP Detection Method
- Procedure: A standard curve for ATP was established using the firefly luciferase bioluminescence assay, based on which the ATP concentrations in samples were calculated to characterize enzyme activity.
- Conclusion: The standard curve proved reliable and is suitable for subsequent comparative analysis of ATP yield and enzyme activity.
We used a firefly luciferase-based ATP detection kit for ATP concentration measurement. Luminescence intensity was measured using a FlexStation®3 multi-mode microplate reader, and data were analyzed with SoftMax Pro software. The ATP concentration in samples was calculated based on the luminescence intensity-ATP concentration standard curve, subsequently reflecting enzyme activity through the ATP concentration. The specific detection method is illustrated in Figure 24.
To calibrate the relationship between ATP concentration and luminescence intensity, we used 20μL of ATP standard solutions at concentrations of 0.1μM, 0.3μM, 1μM, 3μM, and 10μM as samples.Each sample was mixed with 100 μL of ATP detection solution for bioluminescence intensity measurement. After data analysis, a bioluminescence intensity-ATP concentration standard curve was obtained(Figure 25).
3.2 Determining the Ratio of SlPPK-SP to CP-SpyTag in in vivo assembled V-CHARGEs
- Procedure: Integrating modeling results with experimental data, the ratio of SlPPK-SP to CP-SpyTag within in vivo assembled V-CHARGEs was deduced and validated to ensure rigor in subsequent enzyme activity comparisons. (Link to Dry Lab Part2)
- Conclusion: A nearly equivalent ratio of SlPPK-SP to CP-SpyTag within in vivo assembled V-CHARGEs can be achieved by maintaining the total protein concentration at 25%–30% of that used for free SlPPK-SP, thereby ensuring comparability between the assembled system and free enzyme controls in enzyme activity assays.
In the preliminary experiments, based on modeling, we theoretically estimated the encapsulation ratio of SlPPK-SP within in vivo assembled V-CHARGEs. It was determined that when the protein concentration of the in vivo assembled V-CHARGEs sample is controlled at 25%~30% of that of the free SlPPK protein sample, the absolute amount of encapsulated SlPPK-SP is roughly equivalent to that in the free control group. This minimizes interference from differences in protein dosage, ensuring that differences in ATP generation experiments primarily stem from the "free" versus "assembled" states rather than inconsistencies in protein quantity.
Summary
According to the modeling results, to ensure that the amount of SlPPK-SP encapsulated in in vivo assembled V-CHARGEs is essentially the same as that of the free SlPPK-SP control, in subsequent experiments, we controlled the protein concentration of the assembled protein sample to be 25%~30% of that of the free protein sample.
3.3 Prolonged Reaction
- Procedure: The ATP production and reaction rates of free SlPPK-SP and V-CHARGEs were compared over extended reaction periods.
- Conclusion: V-CHARGEs demonstrated a higher and more sustained ATP production capacity during prolonged reactions.
We first measured the changes in ATP content within the system over 6 hours and calculated the rate of ATP content change. The results are shown in Figure 26.
From the ATP generation curve of the prolonged reaction (Figure 26A), the ATP concentration of the control group (free SlPPK) increased faster in the initial phase of the reaction (0~100 min), while the in vivo assembled V-CHARGEs group showed a slightly slower increase rate due to the presence of the assembled system. However, as time progressed (>150 min), the ATP level of the control group plateaued, stabilizing at approximately 1.5 mg/mL, whereas the in vivo assembled V-CHARGEs group continued to rise, eventually maintaining a higher ATP concentration (close to 2.0 mg/mL). This result indicates that although the ATP generation efficiency of the assembled system was slightly lower than the free control in the early stage, it exhibited stronger sustainability and stability under prolonged reaction conditions. Further analysis of the reaction rate changes (Figure 26B) showed that the slope of the control group's curve was significantly higher than that of in vivo assembled V-CHARGEs in the early reaction stage, indicating a faster ATP generation rate. However, over time, the control group's rate decreased rapidly and approached zero, showing gradual decay of its catalytic activity. In contrast, the in vivo assembled V-CHARGEs group, although starting with a slightly lower rate, exhibited a more gradual decline trend, maintaining a certain level of catalytic activity throughout the entire reaction process. This result suggests that in vivo assembled V-CHARGEs can effectively delay the loss of enzyme activity, enabling it to maintain higher ATP yield in prolonged reaction systems.
Summary
The prolonged reaction experiment results show that the initial ATP generation rate of in vivo assembled V-CHARGEs was slightly lower than that of free SlPPK. However, as the reaction time extended, the catalytic activity of the free enzyme rapidly decayed and tended towards inactivation, while in vivo assembled V-CHARGEs still maintained sustained ATP generation capacity. Ultimately, the ATP yield of in vivo assembled V-CHARGEs was higher than that of the control group, and its catalytic rate decline trend was more gradual. This indicates that in vivo assembled V-CHARGEs not only enhance the stability of SlPPK but also extend its functional lifespan in prolonged reaction systems, offering potential advantages for applications in continuous energy supply and long-term industrial reactions.
In practical applications, maintaining enzyme activity over long periods is a key challenge in cell-free synthesis systems and industrial continuous reactions. Free enzymes often rapidly inactivate under prolonged reaction conditions, leading to limited product accumulation and reduced production efficiency. In vivo assembled V-CHARGEs, capable of maintaining stable ATP generation capacity over extended periods, not only provide continuous energy assurance for cell-free synthesis systems but also offer a more reliable power source for industrial fermentation and synthetic reactions requiring long-term operation. This characteristic significantly enhances the potential value of SlPPK in complex environments and large-scale applications.
3.4 Influence of Firefly Luciferase
- Procedure: The influence of varying pH levels, phosphate concentrations, and protease conditions on the luminescence intensity of firefly luciferase in the presence of equivalent ATP was assessed.
- Conclusion: Firefly Luciferase activity remained largely unaffected by changes in pH and phosphate levels, with only minor interference from protease, confirming the robustness of the detection system.
To subsequently assess the effects of factors like pH, phosphate content, and protease content on in vivo assembled V-CHARGEs and free SlPPK, aiming to demonstrate the advantages in vivo assembled of V-CHARGEs under adverse conditions, we first needed to exclude the potential influence of these adverse factors on firefly luciferase activity to ensure experimental rigor. We tested the luminescence intensity generated by equal amounts of ATP activating firefly luciferase under different pH, phosphate concentrations, and protease concentrations, plotted scatter diagrams, and performed linear fitting on the results (Figure 27). We found that firefly luciferase activity was almost unaffected by pH and phosphate, and only slightly affected by protease, validating the reliability of the detection system.
Summary
We found that the luminescence intensity generated by firefly luciferase was essentially the same under different conditions, meaning that firefly luciferase activity was largely unaffected by phosphate concentration and protease, and only slightly affected by pH. However, this minor effect could be mitigated in subsequent calculations through differential methods. This proves the reliability of our experimental results, indicating that the firefly luciferase ATP detection method can be used to assess the effects of pH, phosphate, and protease on SlPPK activity.
3.5 Advantage Assessment
- Procedure: ATP production levels of in vivo assembled V-CHARGEs and free SlPPK were compared under different conditions of pH, phosphate, and protease exposure.
- Conclusion: In vivo assembled V-CHARGEs effectively protected SlPPK from inhibition and degradation, maintaining higher enzymatic activity under various adverse conditions.
To demonstrate the advantages and value of our in vivo assembled V-CHARGEs under unfavorable conditions, we compared the ATP generation levels of in vivo assembled V-CHARGEs and free SlPPK under different phosphate, protease and pH conditions. We found that in vivo assembled V-CHARGEs effectively protected SlPPK from inhibition and degradation, maintaining higher enzyme activity under various adverse conditions.
To investigate the effects of phosphate concentration, protease concentration, and pH on enzyme activity, using free SlPPK as a control, we respectively examined the luminescence intensity after a 15-minute reaction catalyzed by in vivo assembled V-CHARGEs with sodium hexametaphosphate and ADP under different phosphate concentrations, protease concentrations, and pH levels, and generated bar charts for each.
3.5.1 Phosphate
To investigate whether in vivo assembled V-CHARGEs encapsulation could enhance SlPPK's resistance to phosphate inhibition, we detected the relative ATP levels of free SlPPK and in vivo assembled V-CHARGEs-encapsulated SlPPK under different phosphate concentrations (0–20 mM). As shown in Figure 28, under low phosphate concentrations (0–7.5 mM), the ATP generation levels of the two groups were similar, indicating weak phosphate inhibition at this stage. However, under higher phosphate concentrations (≥12.5 mM), the activity of free SlPPK decreased significantly, with relative ATP levels below 0.2. In contrast, SlPPK encapsulated within in vivo assembled V-CHARGEs maintained levels around 0.6–0.8, exhibiting significantly higher residual activity. That is, at phosphate concentrations of 12.5 mM and above, the ATP production level of in vivo assembled V-CHARGEs was significantly higher than that of the control group. These results indicate that in vivo assembled V-CHARGEs encapsulation effectively attenuates the inhibitory effect of phosphate on SlPPK, thereby significantly improving the enzyme's functional stability under adverse conditions.
Summary
The results investigating the effect of different phosphate concentrations on the ATP level of in vivo assembled V-CHARGEs showed that the ATP production activity of in vivo assembled V-CHARGEs under high phosphate treatment was significantly higher than that of the free SlPPK control group, highlighting the superiority of in vivo assembled V-CHARGEs in protecting SlPPK against phosphate.
In practical applications, the presence of high concentrations of inorganic phosphate is a common challenge in many biocatalytic processes. For example, in cell-free synthesis systems, ATP regeneration cycles often lead to the continuous accumulation of phosphate by-products, severely inhibiting the activity of various metabolic enzymes. In industrial-scale fermentation and production processes, buffers and culture media commonly contain high concentrations of phosphate, greatly limiting enzyme stability and reaction efficiency.
In vivo assembled V-CHARGEs encapsulation provides a practical solution to this long-standing bottleneck problem. By protecting SlPPK from phosphate inhibition, in vivo assembled V-CHARGEs not only extend the enzyme's operational lifespan but also maintain high catalytic efficiency in high-phosphate environments, thereby demonstrating higher application value in large-scale production and complex environmental conditions.
3.5.2 Protease
To evaluate whether in vivo assembled V-CHARGEs can protect SlPPK from protease degradation, we compared the residual activity of free SlPPK and in vivo assembled V-CHARGEs-encapsulated SlPPK after treatment with different protease concentrations. As shown in Figure 29, under low concentration protease treatment (20 μg), there was no significant difference in ATP levels between the two groups, indicating that mild protease action was not sufficient to significantly affect enzyme activity. However, as the protease concentration increased, the activity of free SlPPK decreased rapidly, showing a significant reduction in the 40~160 μg range, with less than 40% activity remaining at 160 μg. In contrast, the activity decline of in vivo assembled V-CHARGEs-encapsulated SlPPK was slower, maintaining about 60% activity even under high concentration protease treatment (160 μg). Overall, in vivo assembled V-CHARGEs encapsulation significantly enhanced the stability of SlPPK under protease treatment conditions, further demonstrating its protective effect.
Summary
The results investigating the effect of different protease concentrations on the ATP level of in vivo assembled V-CHARGEs showed that the ATP production activity of in vivo assembled V-CHARGEs under protease treatment was significantly higher than that of the free SlPPK control group, highlighting the superiority of in vivo assembled V-CHARGEs in protecting SlPPK from protease degradation.
In practical applications, exogenous enzymes often reside in complex environments containing proteases, which is almost a "common problem" in industrial applications. Proteases commonly present in industrial fermentation systems can rapidly diminish the activity of added enzymes during scaled-up production, causing a sharp drop in catalytic efficiency. Therefore, resistance to protease degradation is not merely a "tolerance improvement" but addresses a common bottleneck problem spanning industrial, agricultural, and medical biotechnology sectors.
3.5.3 pH
To investigate the effect of acidic environments on SlPPK, we compared the residual activity of free SlPPK and in vivo assembled V-CHARGEs-encapsulated SlPPK under different pH conditions. As shown in Figure 30, at pH 6.5, the ATP generation levels of both groups decreased, with no significant difference observed between them. As the pH decreased to 5.5, the activity of free SlPPK significantly dropped to less than 10%, while in vivo assembled V-CHARGEs-encapsulated SlPPK retained about 20% activity. When the pH was further reduced to 3.5 and 3, the activity of free SlPPK was almost completely lost, whereas in vivo assembled V-CHARGEs-encapsulated SlPPK still maintained levels around 25~30%, displaying significantly higher residual activity. The results show that under different pH conditions, the ATP levels of in vivo assembled V-CHARGEs were generally higher than those of the free SlPPK control group, especially under strongly acidic conditions of pH 3.0 to 3.5, where in vivo assembled V-CHARGEs showed a significant advantage in ATP production activity (***p < 0.001). The results indicate that in vivo assembled V-CHARGEs can effectively protect SlPPK under acidic conditions, significantly enhancing its stability and tolerance.
Summary
The results investigating the effect of different pH on the ATP level of in vivo assembled V-CHARGEs showed that the ATP production activity of in vivo assembled V-CHARGEs under acidic condition treatment was significantly higher than that of the free SlPPK control group, highlighting the superiority of in vivo assembled V-CHARGEs in protecting SlPPK against acidic environments.
In practical applications, extreme pH conditions are a common challenge to enzyme stability and activity. In industrial fermentation and organic acid production processes, systems often become acidic due to the accumulation of metabolic by-products. In food processing, environments like fruit juices and fermented beverages are naturally at low pH. In these scenarios, traditional enzymes often rapidly inactivate, limiting their catalytic applicability.
In vivo assembled V-CHARGEs encapsulation provides an effective solution to this problem. By enhancing the stability and residual activity of SlPPK in acidic environments, in vivo assembled V-CHARGEs not only reduce the need for additional neutralization and regulation steps, thereby lowering production costs, but also broaden the application potential of enzymes in the food industry and environmental remediation, offering a more feasible and sustainable strategy for biocatalysis under extreme conditions.
Overall Summary
Integrating the above results, in vivo assembled V-CHARGEs demonstrated significant protective effects on SlPPK under multiple adverse environmental factors. Whether facing substrate inhibition effects from high phosphate concentrations, degradation risks caused by exogenous proteases, or inactivation issues under acidic conditions, in vivo assembled V-CHARGEs-encapsulated SlPPK performed significantly better than the free enzyme, maintaining higher residual activity and ATP generation capacity. This indicates that in vivo assembled V-CHARGEs not only improve enzyme stability under individual adverse conditions but also provide a universal protective mechanism spanning multiple stress factors.
From a practical application perspective, this "multi-stress resistance" is crucial for the deployment of exogenous enzymes in complex systems. Application scenarios such as industrial fermentation, cell-free synthesis systems, food processing, and environmental remediation often simultaneously present challenges like acidic environments, phosphate accumulation, and protease interference. Traditional free enzymes often rapidly inactivate under these conditions, struggling to maintain catalytic efficiency. In contrast, through its nano-scale encapsulation and protection, in vivo assembled V-CHARGEs not only significantly extend the operational lifespan of SlPPK but also reduce the system's sensitivity to environmental conditions, providing a practical solution for the application of exogenous enzymes under large-scale, long-duration, and extreme conditions.
Overall, in vivo assembled V-CHARGEs exhibit characteristics of a universal platform capable of significantly enhancing enzyme stress resistance. This not only addresses common bottleneck problems in multiple industrial applications but also provides new insights for the future development of biocatalysts and functional materials adapted to extreme environments.
3.6 Discussion
Summary and Reflection
This section systematically compared the catalytic performance of free SlPPK and in vivo assembled V-CHARGEs under prolonged reaction and different stress conditions (phosphate, protease, acidic pH) using a firefly luciferase-based ATP detection kit. Experiments showed that although the initial transient generation rate of the assembled samples was slightly lower than that of the free enzyme, they maintained and accumulated significantly higher ATP levels during prolonged reactions (> 150 min). Under high phosphate concentrations (≥12.5 mM), high protease levels, and acidic conditions (pH < 6.5), in vivo assembled V-CHARGEs consistently exhibited significantly better resistance compared to free SlPPK (statistically significant). This advantage indicates that by providing a protected microenvironment for SlPPK, in vivo assembled V-CHARGEs significantly delay the loss of enzyme activity, allowing it to maintain efficient catalysis even in harsh environments and during prolonged operation. Control experiments with firefly luciferase indicated that the detection system's response to equal amounts of ATP was largely consistent across the tested pH, Pi, and protease ranges, confirming the reliability of the measurements.
These results support the hypothesis that "V-CHARGEs provide a protected microenvironment for SlPPK". This protection may originate from:
1. Spatial segregation and physical protection of the particle shell against proteases, reducing enzyme degradation.
2. Buffering of the local environment (pH, ions) or restricted diffusion of substrates/products leading to slowed enzyme inactivation kinetics.
These findings possess significant scientific value and application prospects. From a scientific perspective, this study clearly demonstrates for the first time that encapsulating polyphosphate kinase using P22-VLP can significantly enhance its tolerance and sustained activity, providing a novel approach to solving the problem of enzyme instability in complex environments. Compared to traditional enzyme immobilization or chemical modification methods, the in vivo assembled V-CHARGEs assembly strategy is more efficient, mild, and biocompatible, substantially improving environmental adaptability while maintaining the enzyme's native activity. From an application perspective, this system shows broad potential in areas requiring long-term, stable energy supply, such as cell-free synthesis systems, in vitro biomanufacturing, and energy metabolism simulation.
Outlook
The enzyme activity assays in this section show that in vivo assembled V-CHARGEs can continuously produce ATP, perform significantly better than free SlPPK under high Pi, protease, and acidic conditions, and maintain higher energy output and stability during prolonged reactions.
Based on these results, our next core objective is to utilize in vivo assembled V-CHARGEs as an energy module, using the ATP they produce to drive enzymatic reactions located on the outer surface of the particle – specifically, by conjugating exogenous enzymes (FLuc, UCK, γ-GCS, GS) to the surface of in vivo assembled V-CHARGEs via SpyTag/SpyCatcher, to validate the feasibility and efficiency of "intra-particle energy supply → extra-particle product synthesis".
4. Product Determination
- By coupling multiple enzymes on the outer surface of V-CHARGEs, we successfully produced 5'-CMP and GSH, demonstrating the platform versatility and high efficiency of V-CHARGEs.
Introduction and Objectives
In the previous experiments, we have verified that SlPPK inside in vivo assembled V-CHARGEs can synthesize ATP, and the product has good enzymatic activity. However, merely proving that in vivo assembled V-CHARGEs can generate ATP inside the shell is not sufficient to demonstrate its potential in practical applications. To truly make it functional, we need to further prove whether this ATP can be efficiently utilized and drive external energy-consuming reactions to produce the desired products.
To this end, we selected three representative ATP-dependent reactions:
- Firefly luciferase luminescence (catalyzed by firefly luciferase)
- 5’-CMP synthesis (catalyzed by Uridine-Cytidine Kinase, UCK)
- Glutathione synthesis (catalyzed in two steps by γ-Glutamylcysteine Synthetase, γ-GCS, and Glutathione Synthetase, GS).
Using SpyTag/SpyCatcher technology to connect them with in vivo assembled V-CHARGEs, the generation of products was detected to demonstrate that in vivo assembled V-CHARGEs can produce ATP to provide energy for external reactions.
4.1 Plasmid construction
4.1 Plasmid construction of FLuc, UCK, γ-GCS, GS
In order to express Firefly Luciferase (FLuc), Uridine-Cytidine Kinase (UCK), γ-Glutamylcysteine Synthetase (γ-GCS), and Glutathione Synthetase (GS) in Escherichia coli BL21 (DE3), we constructed the genetic pathway (Figure 31) and integrated the genes encoding fusion proteins into the plasmid backbone. We chose the pET-28a vector backbone. This T7 promoter-based backbone contains a kanamycin resistance gene as the selection marker and an inducible lac operon (including the lacI repressor), ensuring that IPTG can induce the expression of fusion proteins. In addition, the backbone includes a histidine tag (His-tag) located at the C-terminus of the fusion protein. The presence of this His-tag allows us to subsequently purify the proteins through nickel affinity chromatography. We used SnapGene software to design the plasmids. For pET-28a::SC-UCK, pET-28a::SC-γ-GCS, pET-28a::SC-GS, we used primers synthesized by Sangon Biotech to amplify the target gene fragments, and the gene fragments were inserted into the linearized vector using seamless cloning technology. For the pET-28a::SC-FLuc plasmid, we entrusted Genewiz to synthesize and sequence it, and the results were verified to be correct.
We extracted the plasmids obtained from E. coli DH5α and transformed them into E. coli BL21(DE3) using the heat-shock method. After transformation, the cells were plated on LB medium containing kanamycin for selection. After overnight incubation, we observed different bacterial colonies, indicating successful transformation. Finally, we identified the target genes by Polymerase Chain Reaction (PCR) and agarose gel electrophoresis. From the gel electrophoresis results, the bands corresponding to UCK, γ-GCS, and GS were observed between 1000 bp and 2000 bp (Figure 33), which matched the actual sizes of the gene fragments, proving that the gene fragments had been successfully inserted into the plasmids. We submitted the successfully constructed plasmids to Sangon Biotech for sequencing, and the sequencing results confirmed that the target gene fragments had been successfully integrated into the plasmids.
Summary
In this part, we successfully completed the construction of four recombinant plasmid vectors: pET-28a::SC-FLuc, pET-28a::SC-UCK, pET-28a::SC-γ-GCS, and pET-28a::SC-GS. The plasmids were verified by Polymerase Chain Reaction(PCR) and sequencing, laying a solid foundation for subsequent induced expression and functional studies of FLuc, UCK, γ-GCS, and GS proteins.
4.2 Expression and Purification
To obtain the four proteins, we inoculated overnight seed culture into fresh LB medium at a ratio of 1:100, cultured at 37 ℃ and 200 rpm until OD600 reached 0.6–0.8. Subsequently, 0.1 mM isopropyl-β-D-thiogalactopyranoside (IPTG) was added to induce protein expression, and cultivation was continued at 16 ℃ and 200 rpm for 16 h. The cells were harvested by centrifugation at 6000 rpm for 10 min, resuspended in Buffer A (50 mM Tris-HCl, pH 8.0, 300 mM NaCl, 3 mM β-mercaptoethanol), and disrupted by ultrasonication (3 s sonication/6 s interval cycle, total 10 min). The lysate was centrifuged at 12000 rpm for 45 min, and the supernatant and precipitate were collected separately for subsequent purification.
We used nickel affinity chromatography to separate and purify proteins in the supernatant. First, proteins were eluted with imidazole at different concentration gradients, and then the eluates were subjected to SDS-polyacrylamide gel electrophoresis (SDS-PAGE). Protein expression was analyzed through the gel electrophoresis image (Figure 34). In the lane of SpyCatcher-FLuc, a band of about 100 kDa was observed, which matched the size of SpyCatcher-FLuc and showed almost no impurity bands, indicating that SpyCatcher-FLuc protein purification was successful. In the lane of SpyCatcher-UCK, a band of about 45 kDa was observed, which matched the size of SpyCatcher-UCK and showed almost no impurity bands, indicating that SpyCatcher-UCK protein purification was successful. In the lane of SpyCatcher-γ-GCS, a band of about 75 kDa was observed, which matched the size of SpyCatcher-γ-GCS and showed almost no impurity bands, indicating that SpyCatcher-γ-GCS protein purification was successful. In the lane of SpyCatcher-GS, a band of 45–65 kDa was observed, which matched the size of SpyCatcher-GS and showed almost no impurity bands, indicating that SpyCatcher-GS protein purification was successful.
Summary
In this part, we successfully induced the expression and purified the three proteins FLuc, UCK, γ-GCS, and GS. Subsequently, we will use the SpyTag/SpyCatcher technology to link them with in vivo assembled V-CHARGEs, thereby testing the efficiency of ATP-dependent reactions catalyzed by in vivo assembled V-CHARGEs with externally connected enzymes, and demonstrating the role of in vivo assembled V-CHARGEs in practical production.
4.3 Conjugation, Enzyme Activity Assay, and Characterization
We mixed the purified SpyCatcher-enzyme with in vivo assembled V-CHARGES at a certain ratio, and placed the mixed sample on a shaker or vortex mixer for a certain period of time, allowing enzymes to be conjugated onto the outer surface of in vivo assembled V-CHARGEs. The schematic diagram of the procedure is shown in Figure 35.
4.3.1 FLuc
4.3.1 Firefly Luciferase (FLuc)
We mixed in vivo assembled V-CHARGES after ultracentrifugation with SpyCatcher-FLuc at a mass ratio of 2:1, and shook the mixture at 37 °C for 1 h (specific operation details) to obtain the conjugation product. We performed SDS-PAGE on in vivo assembled V-CHARGES after ultracentrifugation, SpyCatcher-FLuc, and the conjugation product, and analyzed the conjugation through the gel electrophoresis results. The SDS-PAGE result is shown in Figure 36.
Analysis of the gel image reveals that a portion of the V-CHARGEs assembled in vivo were successfully linked to FLuc. However, some residual FLuc remained in the ligation products, indicating that the ligation efficiency was moderate. Therefore, we attempted to purify the ligation products using dialysis.
In order to verify that in vivo assembled V-CHARGEs can provide energy for enzymatic reactions outside the shell, we used Firefly Luciferase (FLuc) as a model enzyme and designed four parallel experiments as follows:
1. No V-CHARGEs and no FLuc group (negative control)
2. With V-CHARGEs but no FLuc group
3. No V-CHARGEs but with FLuc group
4. With V-CHARGEs and with FLuc group (complete system)
All reactions were carried out under the same buffer system and substrate conditions. We used the FlexStation®3 multifunctional microplate reader to detect the bioluminescence intensity produced by firefly luciferase in the four groups, and plotted a bar chart for analysis based on the obtained data (Figure 37).
The detection results showed that in the experimental group with both in vivo assembled V-CHARGEs and FLuc, the bioluminescence signal was significant, indicating that firefly luciferase could efficiently react under this condition. In contrast, the three groups containing only in vivo assembled V-CHARGEs, only FLuc, or the blank control all showed extremely weak bioluminescence signals.
Summary
In this part, we successfully achieved efficient conjugation of SpyCatcher-FLuc with in vivo assembled V-CHARGEs, and obtained conjugates of relatively high purity through dialysis. The bioluminescence detection results of firefly luciferase proved that in vivo assembled V-CHARGEs can provide energy for the external firefly luciferase reaction, thereby supporting its catalytic process. This laid an experimental foundation for subsequently using the energy of in vivo assembled V-CHARGEs to drive the reactions of UCK and γ-GCS/GS, further synthesizing the target products.
4.3.2 UCK
4.3.2 Uridine-Cytidine Kinase (UCK)
We mixed in vivo assembled V-CHARGES after ultracentrifugation with SpyCatcher-UCK at a mass ratio of 2:1, and shook the mixture at 37 °C for 1 h to obtain the conjugation product. We performed SDS-PAGE on in vivo assembled V-CHARGES after ultracentrifugation, SpyCatcher-UCK, and the conjugation product, and analyzed the conjugation through the gel electrophoresis results. The SDS-PAGE result is shown in Figure 38.
Analysis of the gel showed that the band intensity of SpyCatcher-UCK was very close to that of the conjugation product, and almost no extra bands were seen in the conjugation lane, indicating that SpyCatcher-UCK and in vivo assembled V-CHARGES had very high conjugation efficiency and the product was of high purity, achieving the expected conjugation effect.
We characterized the conjugation product using Dynamic Light Scattering (DLS) and further analyzed the shape and particle size using Scanning Electron Microscopy (SEM) of V-CHARGEs(UCK) (Figure 39). DLS results showed two main size distribution peaks, around 20 nm and 100 nm, with particles around 100 nm occupying the dominant intensity proportion, indicating this was the main particle size distribution of the conjugation product. TEM images showed that V-CHARGEs(UCK) presented uniform spherical structures with sizes around 100 nm, consistent with the DLS results.
To test catalytic efficiency, we used High Performance Liquid Chromatography (HPLC) to quantitatively analyze 5′-CMP and cytidine (CR) in the products. Standard curves (Figure 40) were generated to calibrate the peak areas at different concentrations. Figure 41 shows the HPLC chromatograms of the measurement results. The earlier retention time peak corresponds to 5′-CMP, while the later retention time peak corresponds to CR.
Based on the HPLC results, the conjugates successfully produced 5′-CMP with high catalytic efficiency, indicating that our system had good production performance and could provide sufficient ATP to support efficient catalytic reactions for external enzymes.
To demonstrate that in vivo assembled V-CHARGEs could efficiently catalyze reactions in the presence of UCK and play a key role in energy supply, we set up four parallel groups:
1.Without V-CHARGEs and without UCK (negative control)
2.With V-CHARGEs but without UCK
3.Without V-CHARGEs but with UCK
4.With V-CHARGEs and UCK (complete system)
All reactions were carried out under the same buffer system and substrate conditions, with in vivo assembled V-CHARGEs and SpyCatcher-UCK mixed at a mass ratio of 2:1. After the reactions, 5'-CMP concentration was determined by HPLC.
The 5'-CMP yields of the four groups were plotted as a bar chart (Figure 42). Results showed that in the absence of both in vivo assembled V-CHARGEs and UCK, the 5'-CMP concentration was nearly zero. When only in vivo assembled V-CHARGEs or only UCK was added, the 5'-CMP concentration was very low. However, when both in vivo assembled V-CHARGEs and UCK were present, the 5'-CMP concentration significantly increased, reaching the highest value. This confirmed that we successfully synthesized 5'-CMP, and also demonstrated that in vivo assembled V-CHARGEs enhanced reaction efficiency, playing a catalytic role.
Summary
In this part, we successfully conjugated SpyCatcher-UCK with in vivo assembled V-CHARGEs with high efficiency and obtained high-purity products. We characterized the products with DLS and TEM, showing uniform spherical particles with expected size distribution. We analyzed the products with HPLC and designed four parallel experiments, demonstrating the necessity of in vivo assembled V-CHARGEs in the reaction. The results showed that our system had high catalytic efficiency. Overall, our in vivo assembledV-CHARGEs successfully drove energy-consuming reactions after conjugation with UCK and efficiently produced 5′-CMP, suggesting their potential for industrial application.
4.3.3 γ-GCS and GS
4.3.3 γ-Glutamylcysteine Synthetase (γ-GCS) and glutathione Synthetase (GS)
We mixed in vivo assembled V-CHARGES after ultracentrifugation with SpyCatcher-γ-GCS and SpyCatcher-GS at a mass ratio of 30:14:1, and vortexed for 20 min to obtain the conjugation product. We performed SDS-PAGE on in vivo assembled V-CHARGES after ultracentrifugation, SpyCatcher-γ-GCS, SpyCatcher-GS, and the conjugation product, and analyzed the conjugation through the gel electrophoresis results (Figure 43).
Analysis of the gel showed that the conjugation lane had bands corresponding to conjugated V-CHARGEs(γ-GCS/GS) (100–135 kDa), with some unbound γ-GCS and GS visible, and only a small amount of unbound in vivo assembled V-CHARGEs. This indicated that SpyCatcher-γ-GCS and SpyCatcher-GS successfully conjugated with in vivo assembled V-CHARGES with high efficiency, but the mixing ratio could be optimized, as γ-GCS and GS were in excess. To obtain purified products, we used dialysis to remove unbound enzymes.
We characterized the conjugation product using DLS and further analyzed the shape and particle size using Transmission Electron Microscopy (TEM) of V-CHARGEs(γ-GCS/GS) (Figure 44). DLS results showed that the particle size distribution of the conjugates was mainly between 20–100 nm, with particles in the 50–100 nm range dominating. TEM results showed uniform granular structures with sizes consistent with DLS, confirming good morphological characteristics.
After completing the conjugation and characterization of V-CHARGEs(γ-GCS/GS), we further tested the product using glutathione (GSH)-specific ELISA to verify the catalytic efficiency of the conjugated enzymes. To fully evaluate the key role of in vivo assembled V-CHARGEs in catalysis, we designed four parallel groups:
1.Without V-CHARGEs and without γ-GCS/GS (negative control)
2.With V-CHARGEs but without γ-GCS/GS
3.Without V-CHARGEs but with γ-GCS/GS
4.With V-CHARGEs and γ-GCS/GS (complete system)
All reactions were carried out under the same buffer system and substrate conditions, with in vivo assembled V-CHARGEs mixed with SpyCatcher-γ-GCS and SpyCatcher-GS at a mass ratio of 30:14:1. After the reactions, GSH concentration was determined by ELISA.
We first measured a series of GSH solutions with known concentrations using an ELISA kit and generated a standard curve (Figure 45), obtaining the correlation between A450 OD values and GSH concentrations, which served as a reference for subsequent quantification.
A series of standards (100 μg/mL, 50 μg/mL, 25 μg/mL, 12.5 μg/mL, 6.25 μg/mL, 3.125 μg/mL, 1.5625 μg/mL) were measured by ELISA, and the A450 OD values were fitted with a four-parameter logistics equation to generate the GSH ELISA standard curve equation.
We plotted the ELISA results of the four groups using the GSH standard curve (Figure 46). Results showed that in the negative control without in vivo assembled V-CHARGEs and without γ-GCS/GS, the GSH concentration was close to zero; in groups with only V-CHARGEs or only γ-GCS/GS, the GSH concentration was very low, indicating that neither could drive effective production alone. In the complete system with both in vivo assembled V-CHARGEs and γ-GCS/GS, the GSH yield increased significantly, much higher than in the other groups. This demonstrated that only when the energy-supplying system (in vivo assembled V-CHARGEs) and the synthetic enzymes (γ-GCS and GS) were both present could glutathione synthesis be efficiently driven, proving the essential role of in vivo assembled V-CHARGEs as an energy driver.
Summary
In this experiment, we successfully conjugated SpyCatcher-γ-GCS and SpyCatcher-GS with in vivo assembled V-CHARGEs with high efficiency, and characterized the products with SDS-PAGE, DLS, and TEM. Results showed very high conjugation efficiency and uniform spherical particles with size distribution mainly in the 50–100 nm range. Further, by detecting GSH with ELISA and analyzing four control groups, we clearly confirmed that in vivo assembled V-CHARGEs played an indispensable role in γ-GCS and GS-driven glutathione synthesis, providing continuous energy support for efficient catalysis.
Overall Summary
In this section, we successfully achieved external conjugation of UCK and γ-GCS&GS on in vivo assembled V-CHARGEs, and systematically characterized the structure and function of the conjugates. Results showed that UCK, γ-GCS, and GS all conjugated efficiently to the surface of in vivo assembled V-CHARGEs, and the products exhibited uniform and stable particle structures. By detecting products with HPLC and ELISA, we confirmed that both external enzymes catalyzed reactions efficiently under in vivo assembled V-CHARGEs drive, producing target products (5′-CMP and GSH). Control experiments clearly proved the essential energy-supplying role of in vivo assembled V-CHARGEs in these reactions.
These results demonstrate that our in vivo assembled V-CHARGEs can not only efficiently conjugate with multiple enzymes in vitro, but also provide continuous energy for extended reactions, driving complex multi-step metabolic processes. This lays a solid foundation for their application in multi-enzyme cascade reactions, cell-free synthesis systems, and industrial biomanufacturing, highlighting their broad potential in cell factories and synthetic biology applications.
4.4 Discussion
Summary and Reflection
In this section of experiments, we have, for the first time, demonstrated that in vivo assembled V-CHARGEs are not only capable of efficiently synthesizing ATP inside the capsid, but these ATP molecules can also be directly utilized by energy-dependent enzymes attached outside the capsid. By constructing, expressing, and purifying UCK, γ-GCS, and GS, we achieved their efficient connection to in vivo assembled V-CHARGEs using the SpyTag/SpyCatcher system, and successfully completed the in vitro synthesis of 5’-CMP and glutathione (GSH). The experimental results indicate:
1.In the UCK experiments, ATP generated by in vivo assembled V-CHARGEs was able to drive UCK to catalyze the conversion of cytidine to 5’-CMP. HPLC quantitative analysis showed that in the complete system with both in vivo assembled V-CHARGEs and UCK, the yield of 5’-CMP was significantly higher than in the control group, demonstrating the decisive role of ATP provided by in vivo assembled V-CHARGEs in the reaction.
2.In the γ-GCS and GS experiments, ELISA detection revealed that the complete system produced substantial amounts of GSH, whereas the control groups yielded almost no detectable product. This further confirms that in vivo assembled V-CHARGEs can stably supply energy in multi-enzyme cascade reactions, powering complex metabolic pathways.
3.DLS and TEM characterization results both showed that in vivo assembled V-CHARGEs maintained good particle structure and size distribution after enzyme attachment, indicating that the connection process did not significantly compromise their structural stability.
In summary, we successfully verified that in vivo assembled V-CHARGEs can operate in a “energy synthesized inside the capsid, reactions driven outside the capsid” mode, achieving spatially separated ATP transfer and utilization. These findings provide important evidence for the potential of in vivo assembled V-CHARGEs in metabolic engineering and synthetic biology applications.
Prospects
In the future, we plan to further advance this work in the following directions:
1.Expanding product types: Test additional ATP-dependent reactions, such as peptide synthesis, coenzyme regeneration, or production of small-molecule drug precursors, to comprehensively demonstrate the versatility of in vivo assembled V-CHARGEs.
2.Optimizing connection strategies: Improve the efficiency and precision of multi-enzyme cascade reactions by adjusting the number and position of SpyTag/SpyCatcher or employing controllable connection methods.
3.System stability and recyclability: Explore the long-term stability and reusability of in vivo assembled V-CHARGEs, assessing their potential in continuous reactions or industrial reactors
4.Industrial application prospects: Based on our experimental results, in vivo assembled V-CHARGEs could be envisioned as a programmable energy supply platform with broad potential applications in biopharmaceuticals, green synthesis, and environmental remediation.
References
[1]Thuman-Commike, P. A., Greene, B., Malinski, J. A., King, J., & Chiu, W. (1998). Role of the scaffolding protein in P22 procapsid size determination suggested by T = 4 and T = 7 procapsid structures. Biophysical journal, 74(1), 559–568.
[2]Williams, L. A., Neophytou, A., Garmann, R. F., Chakrabarti, D., & Manoharan, V. N. (2024). Effect of coat-protein concentration on the self-assembly of bacteriophage MS2 capsids around RNA. Nanoscale, 16(6), 3121–3132.
[3]Patterson D. P. (2018). Encapsulation of Active Enzymes within Bacteriophage P22 Virus-Like Particles. Methods in molecular biology (Clifton, N.J.), 1798, 11–24.
[4]O'Neil, A., Reichhardt, C., Johnson, B., Prevelige, P. E., & Douglas, T. (2011). Genetically programmed in vivo packaging of protein cargo and its controlled release from bacteriophage P22. Angewandte Chemie (International ed. in English), 50(32), 7425–7428.
[5]Sharma, J., Uchida, M., Miettinen, H. M., & Douglas, T. (2017). Modular interior loading and exterior decoration of a Virus-Like Particle. Nanoscale, 9(29), 10420–10430.
[6]Teschke, C. M., & Parent, K. N. (2010). 'Let the phage do the work': using the phage P22 coat protein structures as a framework to understand its folding and assembly mutants. Virology, 401(2), 119–130.
[7]O'Neil, A., Reichhardt, C., Johnson, B., Prevelige, P. E., & Douglas, T. (2011). Genetically programmed in vivo packaging of protein cargo and its controlled release from bacteriophage P22. Angewandte Chemie (International ed. in English), 50(32), 7425–7428.