Difference between revisions of "Part:BBa K5335001"

Revision as of 08:29, 30 September 2024

The fusion protein of SpyCatcher with EGFP.

Spycatcher can form a covalent connection with Spytag. Using this property, we plan to link Spycatcher with functional proteins so that it can be displayed on the surface of VLP particles in combination with MS2-Spytag.

Usage and Biology

The SpyTag-SpyCatcher system is a fast, reliable and irreversibly linked protein coupling tool developed in recent years, which enables the two to recognize each other with high affinity and spontaneously form isopeptide bonds ^[1]. Taking advantage of this feature, we used a fusion protein composed of SpyCatcher and EGFP to verify whether it could be displayed on the surface of VLP to confirm the reliability of our delivery platform.

Figure 1. The structure of the connection complex formed by SpyTag and SpyCatcher.
The image is from the PDB database with the PDB ID 4MLS. The cyan part represents the SpyCatcher and the orange part represents the SpyTag.

Sequence and Features

Plasmid construction, culture and protein purification validation

Plasmid construction

SpyCatcher-EGFP was constructed on the PUC57 mini plasmid. The upstream MS2-SpyTag is used to verify the binding capability. (Figure 1.)

Figure 2. PUC57 mini plasmid with spycatcher-egfp sequence.

Culture and Purification

We preserved BL21 (DE3) strains that had previously verified the correct sequence introduction. After preliminary experiments to explore the expression conditions, we began to culture the engineered bacteria at a higher amount, and verified the presence of MS2 CP by using the specific binding characteristics of SpyTag-SpyCatcher. We extracted 100 μL of the preserved strain and injected it into 200 mL LB medium for full mixing. The culture bottle was placed in a shaker and cultured at 37℃ and 200 rpm for 8 h until OD₆₀₀. reached about 0.6. The culture bottles were then removed and placed at 16℃ and 160 rpm for 32h. Subsequently, the cultured products were collected at 4℃ and 6000 rpm. After the collected products were washed twice with PBS, the collected bacteria were re-suspended with 8 mL of the bacterial protein extraction kit. 40 μL PMSF was added and 80 μL lysozyme was incubated at 37℃ and 200rpm for 30 minutes. After 30 minutes, the suspension was removed, 20 μL of DNA/RNA enzyme was added, and incubated for another 20 minutes. The suspension was then removed for ultrasonic crushing. The obtained bacterial crushing liquid was centrifuged at 4℃ and 12000 rpm to obtain supernatant and precipitation. The supernatant is separated from the precipitation. The supernatant was temporarily stored at 4℃, and the precipitation was also temporarily stored at 4℃ after being re-suspended with PBS buffer. The protein with 6x His label was purified by Ni-NTA purification column with the supernatant sample. The impurity protein was eluted with a concentration of 10 mM imidazole, and then the tightly bound protein was eluted with a concentration of 250 mM imidazole. We selected the tube with the highest protein concentration from the 250 mM imidazole eluant for SDS-PAGE and Western Blot verification. The antibody used in the Western Blot was directed against the 6x His tag at the carbon terminus of EGFP. The results showed that our target protein existed in the soluble components of the supernatant, and the SpyTag-SpyCatcher connection system could perform its functions correctly. (Figure 3.)

Figure 3. SDS-PAGE and Western Blot of target protein.
A. SDS-PAGE of target protein. 1. Impurity protein eluent a. 2. Impurity protein eluent b. 3. Target protein eluent a. 4. BL21 (DE3) strain. 5. supernatant. 6. precipitate. 7. Target protein eluent b. MW: Molecular weight.
B. Western Blot of target protein. 1.Target protein eluent a. 2.Target protein eluent b. MW: Molecular weight.