Difference between revisions of "Part:BBa K4623001"
| Line 10: | Line 10: | ||
===Characterization=== | ===Characterization=== | ||
We conducted an activity analysis of the expressed mSA(present in the form of sGFP tether<partinfo>BBa_K5301012</partinfo><partinfo>BBa_K5301014</partinfo>). 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 mSA. Observations from Figure 1 indicate that the directly purified soluble sGFP tether exhibits some activity, while the inclusion body proteins, after purification through denaturation and refolding, do not exhibit activity. | We conducted an activity analysis of the expressed mSA(present in the form of sGFP tether<partinfo>BBa_K5301012</partinfo><partinfo>BBa_K5301014</partinfo>). 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 mSA. Observations from Figure 1 indicate that the directly purified soluble sGFP tether exhibits some activity, while the inclusion body proteins, after purification through denaturation and refolding, do not exhibit activity. | ||
| − | <div class="center"><div class="thumb tnone"><div class="thumbinner" style="width:min-content;"><div style="zoom:1. | + | <div class="center"><div class="thumb tnone"><div class="thumbinner" style="width:min-content;"><div style="zoom:1.5;overflow:hidden;"> |
https://static.igem.wiki/teams/5301/parts/elisa.png | https://static.igem.wiki/teams/5301/parts/elisa.png | ||
</div><div class="thumbcaption"> | </div><div class="thumbcaption"> | ||
Revision as of 11:06, 1 October 2024
Monomeric streptavidin(mSA)
Streptavidin is a tetramer protein, and one molecule of streptavidin can bind to tetramolecular biotin with high specificity. The dissociation constant of streptavidin-biotin complex is on the order of 10mol/L, which is a very perfect biotin-binding protein, and its application range is wider than that of avidin, which is also commonly used in biology. Monomeric streptavidin called mSA has been developed by scientists through amino acid mutations and has the highest affinity for biotin among current monovalent streptavidins, with an affinity of 2.8nM[1].
Usage and Biology
Compared to the native recombinant streptavidin tetramer (55 kD), the binding capacity to biotin is equivalent, even the binding effect with biotin-labeled ligands is superior. Overall, it is more effective in avoiding steric hindrance effects due to spatial positioning. Additionally, it can prevent the crosslinking effects caused by tetrameric streptavidin that may lead to aggregation or precipitation, resulting in better experimental reproducibility. The smaller size of mSA allows for easier spatial access to targets, leading to improved labeling efficiency.
Characterization
We conducted an activity analysis of the expressed mSA(present in the form of sGFP tetherBBa_K5301012BBa_K5301014). 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 mSA. Observations from Figure 1 indicate that the directly purified soluble sGFP tether exhibits some activity, while the inclusion body proteins, after purification through denaturation and refolding, do not exhibit activity.
Figure 1.ELISA of the expressed mSA(present in the form of sGFP 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]
References:
[1]Lim KH, Huang H, Pralle A, Park S. Stable, high-affinity streptavidin monomer for protein labeling and monovalent biotin detection. Biotechnol Bioeng. 2013 Jan;110(1):57-67. doi: 10.1002/bit.24605. Epub 2012 Aug 8. PMID: 22806584.