Part:BBa_K3185003

SPYCatcher

Usage and Biology

SpyCatcher is a protein that came from the CnaB2 domain of FbaB, Streptococcus pyogenes. In a natural environment, CnaB2 domain is used for attaching to host cells. In a paper, it is partially changed and divided into two domains.[1] Nowadays, these two protein domains are known as SpyCatcher/SpyTag system because they bind irreversibly with a covalent bond.

In our experiment, we used the SpyCatcher/SpyTag system and designed only SpyCatcher part for assay. In addition, this has two tag or cleavage sites. First is 6×His-tag inserted in the N-terminus of SpyC for protein purification. Second 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 SpyCatcher by using SDS-PAGE.

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  COMPATIBLE WITH RFC[21]
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 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.

References

[1](Peptide tag forming a rapid covalent bond to a protein, through engineered bacterial adhesin)
[2](Programmable polyproteins built using twin peptide superglues)