Difference between revisions of "Part:BBa K3185005"
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Revision as of 11:03, 21 October 2019
SPYCatcher -> CenA W68Y
Usage and Biology
CBM-CenA is a Carbohydrate-binding Module from cellulase of Cellulomonas fimi. Originally, it has binding affinity to cellulose, but its binding ability to PET is enhanced because of point mutation [1]. In the paper, they compare many kinds of mutants and it is found that a mutant whose #68 amino acid W is changed by Y (CenA W68Y) has the most strong binding affinity to PET.
We used it for PET binding domain. We put SpyCatcher on the N-terminus of CenA W68Y because we used SpyCatcher/SpyTag system to bind it to other parts(SpyCatcher:BBa_K1159200, SpyTag:BBa_K1159201). In addition, this has three tag and cleavage sites. First is inserted 6×His-tag in N-terminus of SpyC to purify. Second is inserted MYC-tag between SpyCather and CenA to detect it by using the antibody. Third is inserted TEV protease site because, in the paper, it is used for protein purification. However, we didn’t use it in our experiment.
We put it between BamHI site and Ndel site on pET11-a. The expression plasmids were introduced into BL21(DE3) and expressed by T7 promoter/ T7 RNAP system. Ni-NTA agarose was used for the purification.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal AgeI site found at 751
Illegal AgeI site found at 841 - 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.
Purification
1. E.coli which expressed this part were lysed with sonification.
2. Proteins are purified from lysate with Ni-NTA agarose(QIAGEN).
3. Imidazole eluates were visualized and confirmed by SDS-PAGE followed by CBB staining.
This purification method works. As shown in Fig.1, the protein successfully purified.
Result
Refrences
1
Zhang, Y., Chen, S., Xu, M., Cavoco-Paulo, A.P., Wu, J., and Chen, J. (2010).
Characterization of thermobifida fusca cutinase-carbohydrate-binding module fusion proteins and their potential application in bioscouring∇.
Appl. Environ. Microbiol. 76, 6870–6876.
Zhang, Y., Wang, L., Chen, J., and Wu, J. (2013).
Enhanced activity toward PET by site-directed mutagenesis of Thermobifida fusca cutinase-CBM fusion protein.
Carbohydr. Polym. 97, 124–129.