Part:BBa_K3185007

SPYCatcher -> sfGFP -> TA2

Usage and Biology

Tachystation A2(TA2) is a protein from Tachypleus tridentatus[1]. The paper shows it binds to the polystyrene (PS)[2].

We used it as the PS binding protein. We also inserted Superfolder GFP (sfGFP) which we improved GFP to make the folding time shorter in the N-terminal of LCI (No part name specified with partinfo tag.). By doing so, we wanted to do the binding assay with fluorescence. Moreover, we put SpyCatcher on N-terminus of sfGFP because we used the SpyTag/SpyCatcher system to bind it to other parts.

This part has four tags. First is 6×His-tag inserted in the N-terminus of SpyCatcher for protein purification. Second is MYC-tag inserted between sfGFP and Spy-Catcher to detect it by using the antibody. Third is a TEV protease site and we put it into two regions because it was used for protein purification in the paper[2].

We inserted it in between the BamHI site and the Ndel site on the pET11-a. We used BL21(DE3) for gene expression.We used Ni-NTA agarose for purification. After that, we confirmed the molecular weight of TA2 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
  INCOMPATIBLE WITH RFC[1000]

  Illegal SapI.rc site found at 460
  

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]Horseshoe Crab Hemocyte-derived Antimicrobial Polypeptides, Tachystatins, with Sequence Similarity to Spider Neurotoxins
[2]Directed evolution of polypropylene and polystyrene binding peptides/Programmable polyproteams built using twin peptide superglues