Difference between revisions of "Part:BBa K5335000"

Latest revision as of 01:16, 2 October 2024

MS2 CP tandem dimer fusion containing SpyTag tag

The sequence consists of two MS2 capsid protein subunits in tandem and a SpyTag peptide segment inserted between the two subunits. The protein can correctly self-assemble to form VLP particles of about 22~29nm ^[1], and the Spytag on its surface can combine with Spycatcher to display some functional proteins in the periphery of the particles and become a delivery platform.

Usage and Biology

Plant nematode is one of the important pathogens causing crop diseases in China. It is a serious threat to China's wheat, corn, rice, sweet potato, potato, soybean, vegetables, peanuts, Chinese herbs and other food and economic crops and the safety of production of important diseases. Aiming at nematode control, our project this year selected Virus-like particles (VLPs) as the delivery platform for nematode control functional components. VLPs have significant potential as artificial vaccines and drug delivery systems. We selected MS2 coat protein (MS2 CP) tandem dimer sequences containing SpyTag found in the literature. The protein can self-assemble to form VLP, expose SpyTag sites on the surface, and can specifically bind to functional proteins connected to SpyCatcher to form a functional protein delivery body. The interior can also contain RNA containing specific sequences, which can be used as a transport carrier for gene silencing ^[2].

Sequence and Features

Plasmid construction, culture and protein purification validation

Plasmid construction

The VLP particles we designed belong to constitutive expression in bacteria, so we chose to assemble the MS2 CP fragment on pUC57 mini plasmid. (Figure 1.) After colony PCR and sequencing, we successfully verified the insertion of the correct fragment. (Figure 2.)

Figure 1. Recombinant pUC57 mini plasmid with MS2 CP sequence.

Figure 2. Colony PCR assay of the ms2-spytag fragment.

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 32 h. 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 200 rpm for 30 min. After 30 min, the suspension was removed, 20 μL of DNA/RNA enzyme was added, and incubated for another 20 min. 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 tag 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 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.

Verification of particle assembly

Purification of iodoxanol by density gradient centrifuge particles

In this process, we performed density gradient centrifugation purification of soluble proteins extracted from bacteria. Previous experiments confirmed the expression of the target protein in the bacteria, and the connection of the SpyTag-SpyCatcher system was also verified. Therefore, we cultured 1 L bacterial solution this time, hoping to obtain a larger amount of protein. The process of bacterial culture and protein expression at low temperature was the same as before, followed by ultra-low temperature pressure crushing of the collected bacteria that were re-suspended with PBS buffers. After separating the crushed supernatant and precipitation, the supernatant was precipitated with 3.7 M (NH₄)₂SO₄ for 12 h ^[1], and the precipitation was obtained by centrifugation. PBS buffer was used for re-suspension precipitation, and 0.22 μm filter membrane was used to filter out large particles of the re-suspension ^[2]. Finally, the filtrate is temporarily stored at 4℃.
The samples were then centrifuged at a gradient of 15%, 25%, 40%, 50%, 4℃, 36000 rpm for 4 h. The band obtained by centrifugation was absorbed, and this sample was diluted 1:1 with PBS buffer (V/V) after resuspension. Subsequently, transmission electron microscopy was performed. Correctly assembled VLP particles can be observed.

Figure 4. Density gradient centrifugation results.
The red arrows represent the purified band.

Figure 5. Properly assembled MS2 VLP particles.