Difference between revisions of "Part:BBa K3185010"
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==Purification== | ==Purification== | ||
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<h3><font size="4.5">Expression</font> </h3> | <h3><font size="4.5">Expression</font> </h3> |
Revision as of 09:43, 21 October 2019
SPYCatcher -> engineered PETase
Usage and Biology
Engineered PETase is a protein from Ideonella sakaiensis. The paper tries to improve the binding activity and the degradation activity of PET [1].
We used engineered PETase which shows a higher binding affinity to PET than PETase in order to compare them. We put SpyCatcher(BBa_K1159200) on N-terminus of PETase because we used SpyCatcher/SpyTag system to bind it to other parts.
It has three tags and a cleavage site. First is 6×His-tag inserted in the N-terminus of SpyCather for protein purification. Second is MYC-tag inserted between SpyCatcher and PETase to detect it by using the antibody. Third is a TEV protease site because, in the paper, it was used for protein purification [2]. 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]
- 12INCOMPATIBLE WITH RFC[12]Illegal NheI site found at 751
- 21INCOMPATIBLE WITH RFC[21]Illegal XhoI site found at 1318
- 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal AgeI site found at 1074
- 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 failed. As shown in Fig.1, the protein successfully purified.
Result
References
1 Austin, H.P., Allen, M.D., Donohoe, B.S., Rorrer, N.A., Kearns, F.L., Silveira, R.L., Pollard, B.C., Dominick, G., Duman, R., Omari, K. El, et al. (2018).
Characterization and engineering of a plastic-degrading aromatic polyesterase.
Proc. Natl. Acad. Sci. U. S. A. 115, E4350–E4357.
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.