Part:BBa_K3185010

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].

This time, 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 SpyTag/SpyCatcher 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 CBM (Cellose binding module [[[[[[[[[??more detail??]]]]]]]]] ) 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 the BamHI site and the Ndel site on pET11-a. We used BL21 (DE3) for gene expression. We used the Ni-NTA agarose for purification. After that, we confirmed the molecular weight of EngiPETase by using SDS-PAGE.

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  INCOMPATIBLE WITH RFC[12]

  Illegal NheI site found at 751
  
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal XhoI site found at 1318
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal AgeI site found at 1074
  
• 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.

SDS-PAGE

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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.