Part:BBa_K5335000

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 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].

[1]Peabody DS. Translational repression by bacteriophage MS2 coat protein expressed from a plasmid. A system for genetic analysis of a protein-RNA interaction. J Biol Chem. 1990 Apr 5;265(10):5684-9.

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  COMPATIBLE WITH RFC[21]
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal BsaI site found at 331
  Illegal BsaI site found at 760
  

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.