Difference between revisions of "Part:BBa K3185003"
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<partinfo>BBa_K3185003 short</partinfo> | <partinfo>BBa_K3185003 short</partinfo> | ||
==Usage and Biology== | ==Usage and Biology== | ||
| − | SpyCatcher is a protein that came from the CnaB2 domain of FbaB, <i>Streptococcus pyogenes</i>. 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. | + | SpyCatcher is a protein that came from the CnaB2 domain of FbaB, <i>Streptococcus pyogenes</i>. 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. | Nowadays, these two protein domains are known as SpyCatcher/SpyTag system because they bind irreversibly with a covalent bond. | ||
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| − | 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. | + | 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. |
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| − | ==References== | + | ==References== |
| + | [1](Peptide tag forming a rapid covalent bond to a protein, through engineered bacterial adhesin) | ||
| + | <br> | ||
| + | [2](Programmable polyproteins built using twin peptide superglues) | ||
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===Functional Parameters=== | ===Functional Parameters=== | ||
<partinfo>BBa_K3185003 parameters</partinfo> | <partinfo>BBa_K3185003 parameters</partinfo> | ||
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Revision as of 11:28, 18 October 2019
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
- 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.
SDS-PAGE
References
[1](Peptide tag forming a rapid covalent bond to a protein, through engineered bacterial adhesin)
[2](Programmable polyproteins built using twin peptide superglues)