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, BBa_I746916) which we improved GFP to make the folding time shorter in the N-terminal of LCI (BBa_I746916). 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(BBa_K1159200) 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
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000INCOMPATIBLE WITH RFC[1000]Illegal SapI.rc site found at 460
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.
SDS-PAGE
References
1 Osaki, T., Omotezako, M., Nagayama, R., Hirata, M., Iwanaga, S., Kasahara, J., Hattori, J., Ito, I., Sugiyama, H., and Kawabata, S.I. (1999).
Horseshoe crab hemocyte-derived antimicrobial polypeptides, tachystatins, with sequence similarity to spider neurotoxins.
J. Biol. Chem. 274, 26172–26178.
[2]Directed evolution of polypropylene and polystyrene binding peptides/Programmable polyproteams built using twin peptide superglues
| None |