Difference between revisions of "Team:LMU-TUM Munich/Results"

(Characterization of various Avidin variants)
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==Characterization of various Avidin variants==
 
==Characterization of various Avidin variants==
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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.

Table 1: Properties of various Avidin variants.
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

Literaturreferenz[1]

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Introduction

Design

Experiments

Proof of concept

Demonstrate

Discussion

References

  1. 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.

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