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             <caption>Figure 3: SDS-PAGE of the CBM56-mcherry protein</caption>
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             <caption>Figure 3: SDS-PAGE of the CBM56-mcherry protein.CBM56-mcherry:35.6kD</caption>
 
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Latest revision as of 07:07, 29 September 2024

PbCBM56(CBM56)

Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal NotI site found at 292
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    COMPATIBLE WITH RFC[1000]


BBa_K5530000 - PbCBM56 Profile

Profile

Name: PbCBM56

Gene length: 333 bp

Source: Paenibacillus barengoltzii (6A6C)

Properties:

CBM56 belongs to the carbohydrate-binding module (CBM) family 6. It is an affinity protein for β-1,3-glucan by binding to its non-reducing end, which is a main component of the fungal cell wall [1].

Usage and Biology

CBM56 family proteins are a special class of carbohydrate-binding modules that play an important role in the recognition and hydrolysis of polysaccharides. A study explored the structure and function of BH0236, a multi-module β-1,3-glucanase, which contains a C-terminal module of the CBM56 family. This module binds to soluble β-1,3-glucan laminarin and has a higher affinity for insoluble β-1,3-glucan [2-3].

The role of CBM in polysaccharide recognition, although not specific to CBM56, provides insight into the general application and importance of the CBM family in polysaccharide degradation [2-3]. We used the CBM56 protein to specifically bind to the β-1,3-glucan of the fungal cell wall. This allows the attachment of a probe to the fungal cell wall, and the fungal morphology can be observed through mcherry (Figure 1).

Figure 1: The engineering design schematic
Figure 1: The engineering design schematic

Cultivation, Purification, and SDS-PAGE

Both gene sequences were amplified by PCR, confirmed by bands of approximately 300 bp and 500 bp on the electrophoretic gel. The gene length of CBM56 was 333 bp, indicating successful amplification of the target band. Figure 2 shows that the plasmid was successfully linearized.

Figure 2: The electrophoretic gel map
Figure 2: The electrophoretic gel map

Figure 3 shows the purified protein. The bands corresponding to CBM56-mcherry proteins, ranging from 34 kD to 43 kD, are notably intense, confirming successful expression in the supernatant.

Figure 3: SDS-PAGE of the CBM56-mcherry protein
Figure 3: SDS-PAGE of the CBM56-mcherry protein.CBM56-mcherry:35.6kD

References

[1] Structure of CBM56. Available: http://www.cazy.org/CBM56_structure.html

[2] Cantarel BL, Coutinho PM, Rancurel C, Bernard T, Lombard V, Henrissat B. The carbohydrate-active enzymes database (CAZy): An expert resource for glycogenomics. Nucleic Acids Research, 2009, 37(S1): D233-D238.

[3] Andrew Hettle, Alexander Fillo, Kento Abe, Patricia Massel, Benjamin Pluvinage, David N. Langelaan, Steven P. Smith, Alisdair B. Boraston. Properties of a family 56 carbohydrate-binding module and its role in the recognition and hydrolysis of β-1,3-glucan. Journal of Biological Chemistry, 292, I41, 16955-16968. https://doi.org/10.1074/jbc.M117.806711