Difference between revisions of "Part:BBa K3185003"
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==Purification== | ==Purification== | ||
+ | [[File:SPYCatcher.png|300px|thumb|right|Fig.1 penguin]] | ||
<br> | <br> | ||
<h3><font size="4.5">Expression</font> </h3> | <h3><font size="4.5">Expression</font> </h3> | ||
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<li>Protein was expressed in 0.1mM IPTG for 2hours. | <li>Protein was expressed in 0.1mM IPTG for 2hours. | ||
</ul> | </ul> | ||
− | <h3><font size="4.5"> | + | <h3><font size="4.5">Purification </font></h3> |
− | + | 1. E.coli which expressed this part were lysed with sonification.<br> | |
+ | 2. Proteins are purified from lysate with Ni-NTA agarose(QIAGEN).<br> | ||
+ | 3. Imidazole eluates were visualized and confirmed by SDS-PAGE followed by CBB staining.<br> | ||
+ | <br> | ||
+ | This purification method works. As shown in Fig.1, the protein successfully purified. | ||
<br> | <br> | ||
<br> | <br> | ||
==Result== | ==Result== | ||
− | [[File:191008 Dot Blot.png|300px|thumb| | + | [[File:191008 Dot Blot.png|300px|thumb|right|Fig.2 Plastic-binding protein binding to PET film |
<br> | <br> | ||
A 3µL of protein solution dropped on PET film, then left for 20min. Then the film was washed in TBST for 5min x3, then placed with Anti-His-tag-HRP conjugated for 1h. ECL substrate was added, then chemiluminescence was imaged by LAS-3000. The exposure time is 6min. | A 3µL of protein solution dropped on PET film, then left for 20min. Then the film was washed in TBST for 5min x3, then placed with Anti-His-tag-HRP conjugated for 1h. ECL substrate was added, then chemiluminescence was imaged by LAS-3000. The exposure time is 6min. | ||
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<br> | <br> | ||
<br> | <br> | ||
− | [[File:191001 SPYt-SPYC 2.png|300px|thumb| | + | [[File:191001 SPYt-SPYC 2.png|300px|thumb|right|Fig.3 Isopeptide bond formation between Plastic binding proteins and Encapsulin. |
<br> | <br> | ||
3µL of SpyCatcher-Plastic-binding protein (SpyC-PBP) solution and 3µL of SpyTag inserted TmEncapsulin (SpyTmEnc) solution was mixed, then placed for 16h at room temperature. Then 6µL of 2x SDS sample buffer was added. 10µL of each sample was loaded. SDS-PAGE for 30min in 200V. The gel was CBB stained. | 3µL of SpyCatcher-Plastic-binding protein (SpyC-PBP) solution and 3µL of SpyTag inserted TmEncapsulin (SpyTmEnc) solution was mixed, then placed for 16h at room temperature. Then 6µL of 2x SDS sample buffer was added. 10µL of each sample was loaded. SDS-PAGE for 30min in 200V. The gel was CBB stained. | ||
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<br> | <br> | ||
<br> | <br> | ||
− | [[File:SpyC-T timeD.png|300px|thumb| | + | [[File:SpyC-T timeD.png|300px|thumb|right|Fig.4 Quantification of conjugated band |
<br> | <br> | ||
Conjugated bands’ intensity was quantified with ImageJ. Orange dots show averages value of three experiments. Blacklines show standard deviations. The time point 60min was deleted because it includes negative value. | Conjugated bands’ intensity was quantified with ImageJ. Orange dots show averages value of three experiments. Blacklines show standard deviations. The time point 60min was deleted because it includes negative value. | ||
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<br> | <br> | ||
<br> | <br> | ||
− | [[File:SPYC Time-D graph.png|300px|thumb| | + | [[File:SPYC Time-D graph.png|300px|thumb|right|Fig.4 Quantification of conjugated band |
Conjugated bands’ intensity was quantified with ImageJ. Orange dots show averages value of three experiments. Blacklines show standard deviations. The time point 60min was deleted because it includes negative value. | Conjugated bands’ intensity was quantified with ImageJ. Orange dots show averages value of three experiments. Blacklines show standard deviations. The time point 60min was deleted because it includes negative value. | ||
]] | ]] |
Revision as of 10:05, 21 October 2019
SPYCatcher
Usage and Biology
SpyCatcher is a protein that came from the CnaB2 domain of FbaB, Streptococcus pyogenes(SpyCatcher:BBa_K1159200). 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(SpyCatcher:BBa_K1159200, SpyTag:BBa_K1159201). 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 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]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 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 works. As shown in Fig.1, the protein successfully purified.
Result
![](/wiki/images/f/f1/191008_Dot_Blot.png)
A 3µL of protein solution dropped on PET film, then left for 20min. Then the film was washed in TBST for 5min x3, then placed with Anti-His-tag-HRP conjugated for 1h. ECL substrate was added, then chemiluminescence was imaged by LAS-3000. The exposure time is 6min.
![](/wiki/images/f/f6/191001_SPYt-SPYC_2.png)
3µL of SpyCatcher-Plastic-binding protein (SpyC-PBP) solution and 3µL of SpyTag inserted TmEncapsulin (SpyTmEnc) solution was mixed, then placed for 16h at room temperature. Then 6µL of 2x SDS sample buffer was added. 10µL of each sample was loaded. SDS-PAGE for 30min in 200V. The gel was CBB stained.
References
1 Zakeri, B., Fierer, J.O., Celik, E., Chittock, E.C., Schwarz-Linek, U., Moy, V.T., and Howarth, M. (2012).
Peptide tag forming a rapid covalent bond to a protein, through engineering a bacterial adhesin.
Proc. Natl. Acad. Sci. U. S. A. <i>109.
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.