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
- 10COMPATIBLE WITH RFC[10]
- 12INCOMPATIBLE WITH RFC[12]Unknown
- 21INCOMPATIBLE WITH RFC[21]Unknown
- 23INCOMPATIBLE WITH RFC[23]Unknown
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE WITH RFC[1000]
Purification
Expression
- Cells were grown in 200ml LB media (100μg/ml Ampicillin) at 37oC shaking at 140 rpm to an OD600 of 0.5, verifying via a spectrophotometer.
- Protein was expressed in 0.1mM IPTG for 2hours.
SDS-PAGE
References
[1](Boraston et al. 2004) (Carbohydrate-binding modules: fine-tuning polysaccharide recognition)
[2](Interaction of carbohydrate-binding modules with poly (ethylene terephthalate))
[3](Programmable polyproteins built using twin peptide superglues)
None |