Difference between revisions of "Part:BBa K5335000"

Revision as of 05:42, 30 September 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 ^[1].

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. After colony PCR and sequencing, we successfully verified the insertion of the correct fragment.

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 32h. Subsequently, the cultured products were collected at 4℃ and 6000rpm. 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 10mM imidazole, and then the tightly bound protein was eluted with a concentration of 250mM imidazole. We selected the tube with the highest protein concentration from the 250mM 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. SDS-PAGE and Western Blot of target protein.