Part:BBa_K5301018
sGFP11 tether is composed of mSA, a 3C linker, a 6His tag, and sGFP 11.
sGFP11 tether is composed of mSA (BBa_K4623001), a 3C linker, a 6 His tag, and sGFP11 (BBa_K5301014).We construct membrane protein dimers through the interaction between biotin and streptavidin, as well as the self-assembly mechanism of sGFP.
Figure 1.AlphaFold2 predition of the sGFP11 tether
Usage and Biology
mSA is used for binding to biotinylated proteins, and sGFP1-10 and sGFP11 self-assemble to form GFP, thereby creating a protein dimer that can report the outcome with fluorescence.
Characterization
PCR
We conducted colony PCR validation to assess the feasibility of the plasmid pathway, as shown in Figure 2, where the corresponding bands are very clear, indicating that the target fragments were successfully introduced into the plasmid and transformed into the bacterial strain.
Figure 2.PCR result of the sGFP11 tether.The base sequence length of the sGFP11 tether is 477 base pairs (bp)
SDS-PAGE
SDS-PAGE was used to verify the purification of the sGFP11 tether. Due to the particularity of mSA, we simultaneously purified the soluble proteins from the supernatant and the inclusion body proteins from the pellet after bacterial lysis. It could be observed that the concentration and purity of the purified inclusion body proteins were high, while the concentration of the soluble proteins was low. Further exploration of soluble protein expression or assessment of inclusion body protein activity could be conducted subsequently. Based on the experience with the sGFP1-10 tether, we had purified the soluble and inclusion body proteins of the sGFP11 tether, as depicted in Figure 3.
Figure 3.SDS-PAGE of the soluble sGFP11 tether.The molecular weight of the sGFP11 tether is 17.0 kDa. Bands 4 and 5 represent the elution results of the sGFP11 tether with 100 mM and 150 mM imidazole, respectively. It can be observed that the concentration of the protein eluted with 100 mM imidazole is higher.
ELISA
We conducted an activity analysis of sGFP tethers. Using the standard interaction between streptavidin and biotin as a control, we observed the interaction between the expressed protein and biotin to determine the activity of the expressed sGFP tether. Observations from Figure 4 indicated that the directly purified soluble sGFP tether exhibits some activity, while the inclusion body proteins, after purification through denaturation and refolding, didn't exhibit activity.
Figure 4.ELISA of sGFP11 tether. Row A, wells 1-9 contain a gradient of mSA standard solutions, while wells 10 and 11 are negative controls with 0.5% BSA. Row B, wells 1-10 also contain a gradient of mSA standard solutions, and wells 11 and 12 are negative controls with 0.5% BSA. Row C, wells 2 and 5 are refolded samples of sGFP11 tether protein. Row D, well 2 contains purified samples of sGFP11 tether protein. Rows E and F, well 1 contains purified inclusion body samples of sGFP11 tether.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal AgeI site found at 82
Illegal AgeI site found at 142 - 1000COMPATIBLE WITH RFC[1000]
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