Difference between revisions of "Part:BBa K3185004"

Revision as of 13:24, 17 October 2019

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. (Boraston et al. 2004) (Carbohydrate-binding modules: fine-tuning polysaccharide recognition) In this paper (Interaction of carbohydrate-binding modules with poly (ethylene terephthalate)), 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.

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. (Programmable polyproteins built using twin peptide superglues) 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

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.

SDS-PAGE