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| − | ===Expression of Thaumatin===
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| − | Characterization of this BioBrick was carried out using the yeast expression vector [[Part:BBa_K801004|BBa_K801004]] for transient transfection with the yeast strain INVSc1.
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| − | ==== Ion exchange chromatography ====
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| − | [[Image:TUM12_ThaumatinIEC.png|500px|thump|right|Experimental Results]]
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| − | Preprothaumatin becomes posttranslationally modified by cleaving a part of the N- and the C-terminal polypeptide. Therefore it was not possible to add a tag for affinity chromatography. For this reason it was necessary to purify the protein from the cytoplasm of the disintegrated yeast cells using ion exchange chromatography to have a proof of principle.
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| − | '''Experimental details:'''
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| − | *Samples: cell lysate, supernatant from culture, reference for thaumatin (MedHerbs)
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| − | *Dialysis against 20mM MES Buffer pH 6.0 (twice) using a 12-16 kDa dialysis membrane
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| − | *Chromatography with a Äkta purifier equipped with a Ressource S 6ml (S: Methyl sulfonate (strong cation exchanger))
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| − | *Sample was applied using a super-loop
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| − | *Wash with two column volumes 20mM MES Buffer pH 6.0
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| − | *Elution with a gradient of 0 - 500 mM NaCl over 5 column volumes
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| − | *Fractions of 1 ml were collected during the elution<br>
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| − | '''Experimental results:'''
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| − | *Reference Thaumatin (see figure A and B)
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| − | **nearly no protein in the flow through during sample application (see figure A)
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| − | **a major peak eluting at ~14 mS*cm-1 which corresponds to fraction Nr. 14 see figure A)
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| − | ** The corresponding SDS-PAGE showed a clear band around fraction Nr. 14 corresponding matching the expected 22 kDa
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| − | *Cell lysate
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| − | **high concentration of protein in the flow through (see figure C and D)
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| − | **no clear peak around fraction 14 could be detected
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| − | **The SDS-PAGE performed from the reference and the fractions 12 to 15 showed a weak band at the height of the reference. This band having the same size as the reference (see running properties on SDS-PAGE in figure D) and the same isoelectric point (both eluted in fraction 14) is very likely to be thaumatin which was produced by the yeast cells.
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| − | *Supernatant of yeast culture (see figure E and F)
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| − | **Export of the protein in high concentrations was unlikely to happen, therefore 48 ml of supernatant were loaded on the column
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| − | **Ratio between flowthrough and eluted protein was less favorable compared to the cell lysate.
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| − | **Total protein quantities were to low to be detected by Comassie stain, therefore a silver stain was performed (see figure F)
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| − | **Beside the reference no additional proteinbands could be detected on the silver stained SDS-PAGE<br>
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| − | '''Conclusion from this experiment:'''<br>
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| − | A '''proof of principle for the expression of thaumatin was achieved using ion exchange chromotography and comparison of bands obtained on an SDS-PAGE relative to a standard of thaumatin'''. <br>
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| | ===Applications of BBa_K801080=== | | ===Applications of BBa_K801080=== |
UNIQe78f63311421bfc0-partinfo-00000000-QINU
UNIQe78f63311421bfc0-partinfo-00000001-QINU
