Difference between revisions of "Team:LMU-TUM Munich/Results"
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Streptavidin and its variants were produced by cytoplasmic expression in E. coli in inclusion bodies. After stabilization and refolding of the functional tetrameric and monomeric variants, purification was carried out by ammonium sulfate precipitation, Ion Exchange Chromatography and size exclusion chromatography. Samples of each required step were analysed by SDS-PAGE. | Streptavidin and its variants were produced by cytoplasmic expression in E. coli in inclusion bodies. After stabilization and refolding of the functional tetrameric and monomeric variants, purification was carried out by ammonium sulfate precipitation, Ion Exchange Chromatography and size exclusion chromatography. Samples of each required step were analysed by SDS-PAGE. | ||
Revision as of 20:23, 12 October 2016
Characterization of various Avidin variants
Streptavidin and its variants were produced by cytoplasmic expression in E. coli in inclusion bodies. After stabilization and refolding of the functional tetrameric and monomeric variants, purification was carried out by ammonium sulfate precipitation, Ion Exchange Chromatography and size exclusion chromatography. Samples of each required step were analysed by SDS-PAGE. Finally each of the successfully purified variants were characterized. Mass spectrometry was used to confirm the expected size, circular dichroism spectroscopy to determine folding states, fluorescence titration and surface plasmon resonance for determining binding affinities.
Results can be seen in Table 1.
| Protein | Molar mass [Da] | Isoelectric point (pI) | ε280 [M-1xcm-1] | KD | |
|---|---|---|---|---|---|
| Streptavidin | 52801.36 | 6.09 | 167760 | ||
| Traptavidin | 52516.88 | 5.14 | 167760 | ||
| Streptactin | 52965.76 | 8.32 | 167760 | ||
| Enhanced monomeric avidin | 15220.3 | 5.91 | 35075 | ||
| Single chain avidin | 61530.65 | 9.84 | 94460 |
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Literaturreferenz
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Introduction
Design
Experiments
Proof of concept
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Discussion
References
- ↑ Schmidt, T. G., & Skerra, A. (2007). The Strep-tag system for one-step purification and high-affinity detection or capturing of proteins. Nature protocols, 2(6), 1528-1535.
