CONTENT
Introduction
Summary
Engineering Principle
Construction Process of pET28a-AGE-Linker-NAL
Protein expression
Characterization
Conclusion
References
New improved parts: BBa_25H3L7EW (pET28a-AGE-Linker-NAL)
Existing Part: BBa_K5508004 (AGE)
Based on the existing part BBa_K5508004(AGE), we constructed a new recombinant plasmid BBa_25H3L7EW (pET28a-AGE-Linker-NAL) by inserting a fused AGE-Linker-NAL gene cassette into the pET28a vector via homologous recombination. This fusion design strategically assembles the AGE enzyme, a flexible peptide linker, and the NAL enzyme into a single translational unit, which can significantly enhance the catalytic efficiency of sialic acid synthesis and boost the overall production yield of sialic acid. When AGE and NAL exist as free enzymes, the intermediate product generated by the AGE-catalyzed reaction must reach the active site of NAL through free diffusion in the solution. During this process, the intermediate product may undergo degradation, reversible reactions, and other processes, which ultimately limit the overall reaction efficiency. The linker protein anchors enzymes AGE and NAL within a fixed spatial distance via its flexible or rigid peptide chain structure, forming a molecular channel-like configuration. The intermediate product generated can be rapidly transferred directly to the active site of NAL through the local microenvironment adjacent to the linker. Meanwhile, the linker enables AGE and NAL to freely adjust their conformations in solution, ensuring that the active sites of both enzymes are normally exposed and can bind effectively to their respective substrates. In addition to catalytic efficiency, the fused protein can also improve the overall stability of the enzymes through the synergistic effect of the two enzymes, thereby maintaining high activity under a wider range of reaction conditions and indirectly enhancing the functional performance.
We conducted TLC, ELISA, and HPLC experiments for functional verification. All experimental results confirmed that the catalytic activity of the A-L-N enzyme construct, which was fused with a linker, was higher than that of A-N, which was formed by directly linking the two enzymes.
Advantages compared to the Existing Part (free AGE molecule): During the biosynthetic reaction of sialic acid, AGE solely catalyzes the first step, which involves the epimerization of ManNAc to ManNAc-6P, while NAL exclusively mediates the second step, namely the aldolization of ManNAc-6P to Neu5Ac. Since a single enzyme can only complete a partial step in sialic acid synthesis and cannot independently produce the final product, ligating free AGE alone into the pET28a vector would be insufficient. Therefore, we constructed a new plasmid by ligating AGE, NAL, and a linker into the pET28a vector. This allows the two enzymes (AGE and NAL) to work in combination, enabling the generation of the desired final product, Neu5Ac.
- Spatial proximity and substrate channeling
In contrast to the engineered AGE-Linker-NAL fusion enzyme, the expression of free and soluble AGE and NAL enzymes results in low efficiency for sialic acid production. While the fusion enzyme enables direct substrate channeling, the free enzymes depend on the random diffusion of the reaction intermediate (ManNAc-6P) through the cytoplasm to encounter an NAL enzyme. This inefficient process carries the risk of intermediate loss due to degradation or diversion into competing pathways, thereby limiting the final yield. Conversely, fusing AGE and NAL with a linker facilitates the direct transfer of the intermediate from the active site of AGE to that of NAL. This targeted strategy drastically increases the local concentration of the intermediate at the active site of the second enzyme (NAL), ultimately accelerating the overall conversion of ManNAc to Neu5Ac and significantly enhancing catalytic efficiency compared with the free enzyme system.
- Stoichiometric Balance
The AGE-Linker-NAL fusion construct ensures a fixed 1:1 molar ratio of AGE to NAL, as the two enzymes are translated as a single polypeptide from one transcript. This coordinated expression eliminates metabolic bottlenecks by ensuring efficient consumption of the intermediate product (ManNAc-6P), thereby maximizing the metabolic flux through the sialic acid biosynthetic pathway.
- Simplified Expression and Purification
This fusion strategy significantly streamlines the experimental process. Only a single gene requires cloning, and a single plasmid must be maintained and expressed. Consequently, the entire bifunctional enzyme complex can be purified in one step via nickel-affinity chromatography, leveraging the N-terminal His-tag encoded by the pET28a vector.
The pET28a-AGE-Linker-NAL construct was developed using the pET28a plasmid as its backbone (Figure 1), selected for its suite of critical functional elements: it contains a KanR marker enabling selection on kanamycin-containing media, an IPTG-inducible expression system regulated by the lac operon alongside T7 promoter/terminator sequences, and a His-tag motif to facilitate purification of downstream expressed proteins. These features collectively serve as essential components for the controlled production and isolation of proteins involved in sialic acid biosynthesis.
Figure 1 The plasmid map of pET28a-AGE-Linker-NAL
The construction of the pET28a-AGE-Linker-NAL expression plasmid was initiated by PCR amplification of the AGE-Linker-NAL fusion fragment. Successful amplification was verified via agarose gel electrophoresis, as shown in Figure 2. This revealed a distinct, discrete band corresponding to the expected molecular weight.
Figure 2 Gel electrophoresis validation of pET28a-AGE-Linker-NAL
Concurrently, the pET28a vector was linearized via double digestion using the restriction endonucleases NcoI and XhoI. Successful linearization was confirmed by electrophoretic analysis, as shown in Figure 3.
Figure 3 Double enzyme digestion of plasmid pET28a
We further employed homologous recombination to sequentially assemble the AGE gene, Linker sequence, and NAL gene into the pET28a vector, constructing the pET28a-AGE-Linker-NAL recombinant plasmid. Following the transformation, uniformly distributed single colonies were observed on the agar plate, indicating successful recombinant transformation. Several colonies were randomly selected for colony PCR verification. Electrophoresis results showed a specific band at the expected size (approximately 2303 bp for the AGE-Linker-NAL fragment), preliminarily confirming the correct insertion of the three-gene fusion into the vector. To further validate construction accuracy, positive clone plasmids were extracted for sequencing analysis. Sequence results fully matched the expected sequence, confirming that the AGE, Linker, and NAL elements were correctly integrated into the pET28a vector as designed, with precise codon transitions and no frameshift mutations. These results confirm the successful construction of the pET28a-AGE-Linker-NAL triple fusion expression vector, providing essential material for subsequent studies investigating the regulatory role of the Linker sequence on the spatial conformation and catalytic efficiency of the dual enzymes.
Figure 4 pET28a-AGE-Linker-NAL Plasmid Verification Diagram
Protein expression was induced with IPTG (1 mM) in E. coli BL21(DE3), followed by cell lysis using a sonicator under cold conditions to preserve enzyme activity. The recombinant protein was purified using nickel affinity chromatography via its 6×His-tag. The purification process involved washing with low (buffer A) and high (buffer B) concentrations of imidazole. The collected fractions were analyzed by SDS-PAGE. As shown in Figure 5, a distinct protein band was observed in the elution solution, indicating that our plasmid successfully expresses the target protein.
Figure 5 SDS-PAGE of pET28a-AGE-Linker-NAL
The activities of AGE and NAL were qualitatively analyzed using TLC at two time points: 30 and 60 minutes. As shown in Figure 6, in the AGE-catalyzed reactions, the band corresponding to the non-denatured enzyme at 30 minutes migrated similarly to the product reference, while the denatured enzyme at 60 minutes co-migrated with the substrate. This indicates that 30 minutes was sufficient for optimal AGE activity under these conditions. For NAL, the TLC results showed separation primarily at the substrate positions, suggesting that the current mobile phase may not be ideal for resolving NAL-derived products due to their high polarity.
Figure 6 TLC for AGE and NAL under 30 and 60 minutes
To further validate the generation of ManNAc and NeuAc, we optimized the developing solvent system for thin-layer chromatography (TLC). After reacting at 42°C for 30 minutes, samples underwent two duplicate developments using freshly prepared developing solvent, with TLC plates thoroughly dried using a hair dryer after each development. Subsequently, TLC plates were immersed in a developing agent and heated at 130°C for 5 minutes for visualization.
Results showed that in the three distinct enzyme structures—AGE-NAL (A-N), AGE-Linker-NAL (A-L-N)—no bands corresponding to ManNAc or NeuAc were detected in the A-N group. In contrast, both A-L-N groups exhibited distinct target product bands. This result indicates that introducing Linker or Scaffold modules into the enzyme structure effectively prevents reversible reactions during metabolism, thereby enhancing the synthetic efficiency of ManNAc and NeuAc.
Figure 7 TLC for AGE and NAL under 30 minutes
We conducted systematic TLC analysis of the enzymatic activities of three enzyme constructs—AGE-NAL (A-N), AGE-Linker-NAL (A-L-N)—reacted for one hour at three temperature conditions: 25°C, 37°C, and 42°C. As shown in Figure 8A, the A-N construct yielded the highest amount of ManNAc in the 1-hour incubation group, with no detectable NeuAc product formation at this time point. This indicates that A-N tends to accumulate the intermediate ManNAc over shorter reaction times.
When incubation was extended to 2 hours, as shown in Figure 8B, both the A-L-N constructs exhibited distinct NeuAc bands whose intensity approached that of the target product reference band. This indicates that these constructs efficiently catalyze NeuAc synthesis upon extended reaction time. Compared to the 1-hour incubation group, the 2-hour incubation group exhibited more pronounced product bands at 37°C, indicating that appropriately extending the reaction time significantly enhances catalytic efficiency.
Collectively, these results indicate that the A-N construct favors ManNAc accumulation in short-duration reactions, while the A-L-N constructs promote final NeuAc production after extended reaction times, with 37°C and 2 hours representing optimal conditions. This phenomenon indicates significant differences in catalytic properties among different enzyme constructs. The introduction of linkers or scaffolds may promote the conversion of reaction intermediates into end products, thereby enhancing overall catalytic efficiency.
Figure 8 TLC for NAL-AGE (A-N), AGE-Linker-NAL (A-L-N) under 25°C, 37°C, and 42°C for 1 hour,and 2 h
To evaluate the catalytic performance of the dual-enzyme system, an ELISA-based quantification assay was conducted.
Table 1 The standard table of Neu5Ac
|
Concentration (ng/mL) |
Absorbance |
|
0 |
0.0889 |
|
10 |
0.5624 |
|
20 |
1.0481 |
|
40 |
1.5943 |
|
80 |
2.0665 |
|
160 |
2.3355 |
A 4-parameter logistic regression (4PL) model was employed for standard curve fitting, with the equation shown in Figure 9, which exhibited a high correlation coefficient (R² = 0.9999).
Figure 9 The equation and the standard curve for the ELISA experiment
The absorbance measured in the experiment was substituted into the equation for calculation to obtain the initial detected concentration of sialic acid. The actual content of sialic acid in the samples was then derived by multiplying by the dilution factor (5 times), as shown in Table 2.
Table 2
|
Samples |
Absorbance |
Initial detection concentration |
Actual concentration |
|
Control Group |
0.0457 |
0.0000 |
0.0000 |
|
1.6396 |
41.9429 |
209.7146 |
|
|
A-N 5 mg/mL |
0.2220 |
3.4540 |
17.2700 |
|
A-N 10 mg/mL |
0.4331 |
7.4243 |
37.1216 |
|
A-L-N 1 mg/mL |
0.6802 |
12.1123 |
60.5615 |
|
A-L-N 5 mg/mL |
0.7649 |
13.8322 |
69.1611 |
|
A-L-N 10 mg/mL |
1.0244 |
19.7337 |
98.6683 |
|
S+A-N 1 mg/mL |
1.2331 |
25.5186 |
127.5932 |
|
S+A-N 5 mg/mL |
0.8622 |
15.9175 |
79.5877 |
|
S+A-N 10 mg/mL |
1.4269 |
32.2130 |
161.0652 |
The final results were plotted in Figure 10. For the enzyme construct A-N, the sialic acid synthesis efficiency was the highest at a concentration of 1 mg/mL, and decreased at medium to high concentrations (5-10 mg/mL). At a low concentration, the rate of the first-step reaction in sialic acid synthesis was slightly higher than that of the second-step reaction, resulting in the overall reaction proceeding toward sialic acid synthesis. Under high concentration conditions, the product sialic acid was decomposed into intermediate products by the NAL enzyme, which were further decomposed by the AGE enzyme.
For the enzyme construct A-L-N, the content of sialic acid showed an upward trend as the concentration increased. Compared with the maximum value of 1 mg/mL in the A-N group, the detection values at various concentrations in the A-L-N group were relatively stable. This indicated that the role of the Linker is to stabilize enzyme function rather than overactivate it.
Figure 10 Concentration of Neu5Ac tested by ELISA Kit calculated by the standard curve
The catalytic experiment of the A-N enzyme construct was illustrated in Figure 11A. The sialic acid content reached its highest level at a reaction time of 60 minutes across all three reaction temperatures. As the reaction time prolonged, the sialic acid yield exhibited a decreasing trend. We hypothesize that this phenomenon was associated with the reversibility of the catalytic reaction. When the content of the intermediate product ManNAc-6P accumulated to a certain extent, the reaction proceeded in the reverse direction. Consequently, the substrate for the second step of the reaction was reduced, leading to a decrease in the final yield of sialic acid. Under the condition of a reaction time of 60 minutes, the reaction temperature of 42 °C yielded the highest accumulation of sialic acid. Overall, the optimal reaction system for enzyme construct A-N is as follows: temperature 42 °C + reaction time 60 minutes.
The similar results were also observed when enzyme constructs A-L-N were used, as shown in Figure 11B. Overall, the optimal reaction systems for both enzyme constructs A-L-N were as follows: temperature 42 °C + reaction time 60 minutes.
Figure 11 Sialic acid production by each enzyme construct under different reaction times and temperatures
Through detailed analysis of the HPLC peak chart in Figure 12, the successful generation of sialic acid and the significant catalytic advantage of the A-L-N construct can be further confirmed.
In the chromatogram of the A-L-N construct, a distinct and sharp peak appears at approximately 11 minutes retention time, which aligns perfectly with the characteristic peak of sialic acid in the standard sample. This peak exhibits symmetry and a large area, indicating high product concentration and excellent separation with no significant tailing.
In contrast, although product peaks are detectable at the same retention time for other constructs (e.g., A-N), their response intensity is significantly lower than that of A-L-N. These peaks are smaller and accompanied by minor impurity peaks. Particularly in the A-N sample, a chromatographic peak belonging to the intermediate ManNAc (retention time approximately 3 minutes) appeared before the target product peak, further indicating incomplete reaction and lower catalytic efficiency.
In summary, quantitative HPLC analysis combined with prior qualitative TLC results collectively demonstrate that the A-L-N construct is the most efficient catalytic module. The synergistic enzyme activity achieved through its linker sequence significantly enhances both sialic acid yield and purity, outperforming the A-N construct.
Figure 12 Chromatograms of sialic acid content under 42 ℃, 240 minutes
Figure 13 shows the sialic acid yield produced by the two plasmids at 42°C for 120 minutes. Although sialic acid content gradually decreased with extended reaction time, the decline was minimal for the A-L-N enzyme assembly, which achieved the highest final cumulative product concentration. This indicates that the A-L-N construct effectively reduces reversible reactions, making it the optimal enzyme architecture for neuraminic acid production.
Figure 13 Sialic acid content under 42 ℃, 120 minutes
Compared with the basic A-N construct, the A-L-N fusion protein demonstrates markedly superior stability and catalytic efficiency. This enhancement is primarily attributable to the engineered linker, which functions as a sophisticated molecular bridge. This bridge strategically integrates the AGE and NAL enzymes into a unified molecular machine, effectively addressing three critical limitations inherent in free enzyme systems: inefficient substrate transfer, mutual conformational interference, and a high propensity for undesired side reactions. By spatially organizing the two catalytic domains, the linker facilitates direct substrate channeling, minimizes steric clashes, and creates a protected micro-environment, thereby transforming two separate enzymes into a synergistically functioning unit that significantly boosts the overall catalytic efficiency.
