Part:BBa_K3185004

SPYCatcher -> BaCBM2

Usage and Biology

BaCBM2 is a Carbohydrate-Binding Module (CBM) from Bacillus anthracis. CBM often found in Carbohydrate related enzymes. It can bind to not only highly crystallized cellulose but also PET because it has a binding site formed by aromatic amino acids[1].In this paper, they research binding affinity of some kinds of CBM and PET. As a result, it is found that BaCBM2 has the most strong binding affinity to PET[2].

We used BaCBM2 as PET binding domain. We put SpyCatcher on N-terminus of BaCBM2 because we used SpyTag/SpyCatcher system to bind it to other parts. Also, this has three tag and cleavage sites. First is 6×His-tag inserted in the N-terminus of SpyC for protein purification. Second is MYC-tag inserted between SpyC and CBM to detect it by using the antibody. Third is a TEV protease site because, in the paper, it was used for protein purification[3]. However, we didn’t use it in our experiment.

We put it between BamHI site and Ndel site on pET11-a. We used BL21 (DE3) for gene expression. We used Ni-NTA Agarose for purification. After that, we confirmed molecular weight of BaCBM2 by using SDS-PAGE.

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
  COMPATIBLE WITH RFC[1000]

Purification

Expression

• Cells were grown in 200ml LB media (100μg/ml Ampicillin) at 37^oC shaking at 140 rpm to an OD600 of 0.5, verifying via a spectrophotometer.

• Protein was expressed in 0.1mM IPTG for 2hours.

References

1 Boraston, A.B., Bolam, D.N., Gilbert, H.J., and Davies, G.J. (2004). Carbohydrate-binding modules: Fine-tuning polysaccharide recognition. Biochem. J. 382, 769–781.

2 Veggiani, G., Nakamura, T., Brenner, M.D., Gayet, R. V., Yan, J., Robinson, C. V., and Howarth, M. (2016). Programmable polyproteams built using twin peptide superglues. Proc. Natl. Acad. Sci. U. S. A. 113, 1202–1207.

3 Weber, J., Petrović, D., Strodel, B., Smits, S.H.J., Kolkenbrock, S., Leggewie, C., and Jaeger, K.E. (2019). Interaction of carbohydrate-binding modules with poly(ethylene terephthalate). Appl. Microbiol. Biotechnol. 103, 4801–4812.