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Revision as of 00:56, 19 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 (cd) to determine folding states, fluorescence titration and surface plasmon resonance for determining binding affinities.
Results can be seen in Table 1.
All cd spectra were evaluated by Spectra Manager Software and results can be seen in figure 1.
protein | amino acids (without Met) | molecular weight monomer [Da] | molecular weight tetramer [Da] | theoretical isoelectric proint (pI) | pI (Isoelectric focusing) | extinction coefficient (monomer, ε280) [M-1 cm-1] | KD |
---|---|---|---|---|---|---|---|
Streptavidin wt | 126 | 13200.34 | 52801.36 | 6.09 | 41940 | ||
Traptavidin | 126 | 13129.22 | 52516.88 | 5.14 | 41940 | ||
Streptactin | 126 | 13241.44 | 52516.88 | 8.32 | 41940 | ||
enhanced monomeric Avidin | 138 | 15220.30 | / | 5.91 | 35075 |
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Introduction
Design
Experiments
Proof of concept
Demonstrate
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.