Part:BBa_K5335025
SpyCatcher-VDAL-CPPs (R9-Tag)
To enhance plant immunity, a novel fusion protein, SpyCatcher-VDAL-CPPs, was designed. This construct incorporates a SpyCatcher tag[1] for conjugation, VDAL for immune activation[2], and the R9 cell-penetrating peptide for intracellular delivery[3]. Functional studies demonstrated the capacity of this fusion protein to penetrate plant cell membranes and induce an ETI response. Molecular characterization and DAB staining confirmed the successful construction and immunogenic activity of the fusion protein. The design was verified as shown in Figure 1.
Usage and Biology
VDAL, an Aspf2-like protein derived from Verticillium dahliae, has been shown to activate PTI responses when expressed extracellularly [4] and can also induce endogenous ETI immune responses when expressed intracellularly. To verify the function of the constructed circuit, a pET28a-based expression system was employed, with the T7 promoter under the control of the lac operon. The circuit includes the engineered SpyCatcher-VDAL-CPPs protein, SpyTag-6*His protein, and SpyCatcher-CPPs protein. The SpyCatcher-SpyTag (SpyC/SpyT) system is derived from the CnaB2 domain of the Streptococcus pyogenes fibronectin-binding protein. SpyC is an immunoglobulin-like protein with a molecular weight of approximately 12 kDa, while SpyT is a short peptide consisting of 13 residues. These two components can specifically recognize each other and spontaneously form an isopeptide bond, enabling high-affinity binding. The designers aimed to utilize this covalent linkage during co-expression to verify the functionality of the SpyCatcher-SpyTag system and facilitate the purification of the target protein. The constructed plasmid vector is illustrated in Figure 2.
Experimental Verification
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE WITH RFC[1000]
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