Difference between revisions of "Part:BBa K5396000"

Revision as of 19:30, 20 September 2024

BaCBM2_RFP_3xMad10

This CBM2 protein is fused with the red fluorescent protein (RFP), which exhibits an excitation maximum at 558 nm and an emission maximum at 583 nm. This fusion enhances the visualization of CBM2. The protein also has three MAD10 peptides [ ], which serve as a magnetic tag that facilitates the purification of the protein through magnetic separation techniques.

This part was used as template to construct BBa_K5396003.

Usage and Biology

This CBM2, or Carbohydrate-Binding Module 2, is a protein sourced from Bacillus anthracis. It belongs to a broader family of carbohydrate-binding modules that are crucial for the degradation of polysaccharides. These modules are important to break down complex carbohydrates, enabling microorganisms to convert them into usable energy sources.

Recent study [ ] has shown that CBM2 has the ability to bind to certain types of plastics, especially those derived exhibiting similar structural features of polysaccharides. This binding ability is largely due to the protein's carbohydrate-binding properties, which facilitate interactions with specific functional groups found on plastic surfaces.

Figure 1. AlphaFold 3 3D simulation of BaCBM2 with miRFP and three Mad10 tags.

Characterization

SEC-MALS

Knowing that the protein 6His-CBM-RFP-3xMAD10 has a 44.86 kDa molecular mass (MM), it is possible to observe three different oligomeric states in the SEC-MALS result shown in Figure 2. With a smaller normalized signal (dRI), it was possible to observe a MM of 180±2 kDa around 32 minutes of elution, which is close to the molecular mass of a 4 units oligomer.

Following on, a second elution was made around 34 minutes with 86.2±0.5 kDa. According to this molecular mass, it is possible to observe a dimer structure of the protein with a higher dRI. Finally, with the highest dRI and a molecular mass of 46.1±0.2 kDa at 37 minutes of elution, it was possible to identify the protein monomer state.

When compared to the other oligomer states, it was notable that this last elution had the lowest standard deviation when compared to the others. As a conclusion, it is possible to confirm that the 6His-CBM-RFP-3xMAD10 construction is most likely to be a single unit protein.

Figure 2. SEC-MALS result of 6His-CBM-RFP-3xMAD10.

Circular Dichroism (CD)

If you want to check the similarities between the BaCBM2 and BARBIE1, see our results page.

Sequence and Features