Part:BBa_K4766009
SpyCatcher-Mi3
In our project, we aim to ascertain if the lab-engineered protein cage, Mi3, can serve as a universal detection tool for the rapid detection of various disease markers, inflammatory factors, or environmental pollutants. After fusion-coupling tags to the Mi3 subunits, they can assemble with cargo proteins that also bear the same coupling tags. When expressed, the 60 Mi3 subunits can spontaneously assemble into an icosahedral protein cage, Mi3. Leveraging the 60 sites on the protein cage, we can illuminate the protein cage by introducing a high ratio of eGFP fluorescent reporter molecules. Furthermore, by introducing a specific antibody molecule at a certain ratio, we utilize immunorecognition to capture biological markers, thereby achieving efficient detection of specific biological molecules.
The expression of SpyCatcher-Mi3 protein
Upon obtaining the amino acid sequences for Mi3 and SpyCatcher, we utilized the pET28a vector to construct the recombinant plasmid pET28a-SpyCatcher-Mi3 (as shown in Figure 1). Using PCR amplification, we obtained the target fragment containing SpyCatcher-Mi3 from the plasmid pET28a-SpyCatcher-Mi3 (as depicted in Figure 2), validating the successful construction of the recombinant protein plasmid.
Fig.1 Design diagram of the target protein gene segments for the recombinant plasmids pET28a-SpyCatcher-Mi3.
Fig.2 PCR fragment electrophoresis image of the recombinant plasmid pET28a-SpyCatcher-mi3
Subsequently, using this recombinant protein plasmid, we successfully expressed the soluble protein SpyCatcher-Mi3 (as shown in Figure 3).
Fig.3 Lane 2: resuspended pellet from E. coli BL21 (DE3) cell lysis. Lane 3: supernatant from E. coli BL21 (DE3) cell lysis. Lane 5,6,7,8: Elution Buffer after Ni-NTA purification
Hence, in this project, we selected the SpyCatcher/SpyTag system to achieve the loading of the target protein onto Mi3 Biological coupling system reaction Mi3-eGFP In our project, SpyCatcher-Mi3 and SpyTag-eGFP were assembled in different proportions. As shown in Figure 4, the gray value analysis showed that when the feeding ratio of SpyTag-EGFP to SpyCatcher-Mi3 was 3:2, the assembly efficiency was the highest, reaching 63.57%, as shown in Table 1.
Fig.4 Lanes 2, 3, 4, 5 represent samples assembled with Mi3 and eGFP in the ratios of 1:1, 6:5, 3:2, 2:1, 3:1, and 6:1, respectively. Lane 8 serves as the control group with known concentrations of SpyCatcher-Mi3.
Table 1. Assembly Efficiency of Samples Assembled in Ratios of 1:1, 6:5, 3:2, 2:1, 3:1, and 6:1 for SpyTag-eGFP and SpyCatcher-Mi3.
Therefore, this project chose the SpyCatcher-SpyTag bioconjugation system to link eGFP with the protein cage Mi3. The assembly efficiency is maximized when the molar ratio is 3:2 for SpyTag-eGFP and SpyCatcher-Mi3
Introduction of SpyTag-Streptavidin and Optimization of Assembly Ratio.<\h3>
Initially, we assembled SpyCatcher-Mi3 and SpyTag-eGFP to preliminarily investigate the ratio when system's fluorescence value could reach its peak. Based on the fluorescence standard curve drawn from the preliminary exploration(as shown in Fig. 5), we further examined the precise assembly ratio of the three proteins around the proportion with the highest fluorescence value.
We characterized the assembly samples of the three proteins using fluorescence and nanoflow cytometry.
(1) Fluorescence Characterization: We set up six proportions for the assembly of SpyCatcher-Mi3 subunits and SpyTag-eGFP. After the assembly was completed, we performed dialysis to remove unassembled proteins.
Fig. 5 Fluorescence value standard curve for SpyCatcher-Mi3 Subunit and SpyTag-eGFP assembly samples.
(2) Nanoflow Cytometry Characterization: We have found that when the assembly ratio of SpyCatcher-Mi3 subunits to SpyTag-eGFP is around 60:50, the fluorescence value of the assembled sample reaches maximum(as shown in Fig. 5). To achieve the maximum signal amplification ratio, we aim to allow the protein cage to bind as many SpyTag-eGFP molecules as possible. Based on this, we set up three ratios for SpyCatcher-Mi3 subunits to SpyTag-Streptavidin, which are 60:5, 60:10, and 60:15. After adding biotin-PEG-FITC to the system and performing extensive dialysis, we conducted nanoflow cytometry characterization, and the results obtained are as follows.(as shown in Fig. 6)
Fig. 6 Nanoflow cytometry characterization of the assembly with a ratio of SpyCatcher-Mi3 to SpyTag-Streptavidin-FITC at 60:5, 60:10, 60:15.
As we increase the amount of SpyTag-Streptavidin-FITC added to the system, the fluorescence intensity of the protein cage correspondingly rises. This indicates that under this assembly ratio, SpyCatcher-Mi3 and SpyTag-Streptavidin can successfully assemble, and it's positively correlated with the added SA concentration. Subsequently, based on the previous results, we set two ratios: SpyCatcher-Mi3 subunit: SpyTag-eGFP: SpyTag-Streptavidin at 60:55:5 and 60:50:10, to investigate the ratio that can achieve the maximum fluorescence value. The results obtained are as follows.(as shown in Fig. 7)
Fig. 7 a) Nanoflow cytometry characterization of the assembly with the ratio of SpyCatcher-Mi3 subunit, SpyTag-eGFP, and SpyTag-Streptavidin at 60:50:10. b) Nanoflow cytometry characterization of the assembly with the ratio of SpyCatcher-Mi3 subunit, SpyTag-eGFP, and SpyTag-Streptavidin at 60:55:5.
When the assembly ratio of SpyCatcher-Mi3 subunit, SpyTag-eGFP, and SpyTag-Streptavidin is 60:50:10, the fluorescence intensity is the highest and the fluorescence distribution is uniform. However, when the ratio is 60:55:5, the fluorescence intensity is slightly lower than the former and the fluorescence distribution is more dispersed.Molecular Affinity Measurement.<\3>
To verify the effectiveness of our constructed monitoring system, we need to measure the efficiency with which the antibody, loaded onto the protein cage, captures the target antigen. However, the concentration of the samples is relatively low, and the measurement of protein sample-related parameters has high environmental requirements. Therefore, we chose microcalorimetry as an appropriate method to detect this parameter.
Fig.8 Fluorescence MST (Microscale Thermophoresis) curve of the assembled sample.
We constructed a modular protein system with a stoichiometry of SpyCatcher-Mi3 subunits: SpyTag-eGFP:SpyTag-Streptavidin = 60:50:10. We selected the commonly used BSA antigen and antibody to validate the affinity of the system. To ensure the reliability of the experimental data, We set up 16 gradient dilutions for affinity determination.
Fig.9 The affinity constant curve of IgG to BSA on the assembled protein cage.
Through the ITC (Isothermal Titration Calorimetry) verification of the protein system we constructed, we concluded the following two points: (1) The MST (Microscale Thermophoresis) curve has a good overlap, which proves that the fluorescence of the assembled Target sample (Mi3-EGFP-SA-IgG) is uniform. (2) The affinity constant Kd of IgG on the assembled protein cage to BSA is 53.7 nM, which is slightly higher than the common affinity between BSA and its antibody. The uniformity of the fluorescence signal and the relatively high affinity constant both indicate that the protein system we constructed can efficiently capture the target antigen and transmit a stable fluorescence intensity, providing a foundation for our subsequent semi-quantitative paper-based detection.Characterization<\h4>
In a 1×PBS buffer solution, characterized by Dynamic Light Scattering (DLS), we observed that the average particle size of SpyCatcher-Mi3 increased to approximately 2000 nm at 45℃ (as shown in Figure 10). Thus, we hypothesize that the SpyCatcher-Mi3 protein undergoes denaturation in the temperature range of 35-50℃.
Fig. 10 Variation in the average particle size of SpyCatcher-Mi3 with temperature.
Additionally, data from DLS suggests that SpyCatcher-Mi3 may undergo aggregation (Figure 11).
Fig. 11 Particle size distribution of SpyCatcher-Mi3 at various temperatures.
Sequence and Features| None |