Difference between revisions of "Part:BBa K801080:Experience"

(→‎Ion exchange chromatography)
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==== Ion exchange chromatography ====
 
==== Ion exchange chromatography ====
 
[[Image:TUM12_ThaumatinIEC.png|500px|thump|right|Experimental Results]]
 
[[Image:TUM12_ThaumatinIEC.png|500px|thump|right|Experimental Results]]
−
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 desintegrated yeast cells using ion exchange chromatography to have a proove of principle.
+
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.
  
 
'''Experimental details:'''
 
'''Experimental details:'''
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*Chromatography with a Äkta purifier equipped with a Ressource S 6ml (S: Methyl sulfonate (strong cation exchanger))
 
*Chromatography with a Äkta purifier equipped with a Ressource S 6ml (S: Methyl sulfonate (strong cation exchanger))
 
*Sample was applied using a super-loop
 
*Sample was applied using a super-loop
−
*Wash with two colum volumes 20mM MES Buffer pH 6.0
+
*Wash with two column volumes 20mM MES Buffer pH 6.0
 
*Elution with a gradient of 0 - 500 mM NaCl over 5 column volumes
 
*Elution with a gradient of 0 - 500 mM NaCl over 5 column volumes
 
*Fractions of 1 ml were collected during the elution<br>
 
*Fractions of 1 ml were collected during the elution<br>
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'''Experimental results:'''
 
'''Experimental results:'''
 
*Reference Thaumatin (see figure A and B)
 
*Reference Thaumatin (see figure A and B)
−
**nealy no protein in the flow through during sample application (see figure A)
+
**nearly no protein in the flow through during sample application (see figure A)
 
**a major peak eluting at ~14 mS*cm-1 which corresponds to fraction Nr. 14 see figure A)
 
**a major peak eluting at ~14 mS*cm-1 which corresponds to fraction Nr. 14 see figure A)
 
** The corresponding SDS-PAGE showed a clear band around fraction Nr. 14 corresponding matching the expected 22 kDa
 
** The corresponding SDS-PAGE showed a clear band around fraction Nr. 14 corresponding matching the expected 22 kDa
 
*Cell lysate
 
*Cell lysate
 
**high concentration of protein in the flow through (see figure C and D)
 
**high concentration of protein in the flow through (see figure C and D)
−
**no clear peak arround fraction 14 could be detected  
+
**no clear peak around fraction 14 could be detected  
−
**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 propperties 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.
+
**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.
 
*Supernatant of yeast culture (see figure E and F)
 
*Supernatant of yeast culture (see figure E and F)
 
**Export of the protein in high concentrations was unlikely to happen, therefore 48 ml of supernatant were loaded on the column
 
**Export of the protein in high concentrations was unlikely to happen, therefore 48 ml of supernatant were loaded on the column
−
**Ratio between frowthrough and eluted protein was less favourable compared to the cell lysate.  
+
**Ratio between flowthrough and eluted protein was less favorable compared to the cell lysate.  
−
**Total protein quantities were to low to be detected by Coomassie stain, therefore a silver stain was performed (see figure F)
+
**Total protein quantities were to low to be detected by Comassie stain, therefore a silver stain was performed (see figure F)
 
**Beside the reference no additional proteinbands could be detected on the silver stained SDS-PAGE<br>
 
**Beside the reference no additional proteinbands could be detected on the silver stained SDS-PAGE<br>
  
 
'''Conclusion from this experiment:'''<br>
 
'''Conclusion from this experiment:'''<br>
−
A '''proof of principle for the expression of Thaumatin was achieved using ion exchange chromotography and comparison of bands optained on an SDS-PAGE relative to a standard of Thaumatin'''. <br>
+
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>
  
 
===Applications of BBa_K801080===
 
===Applications of BBa_K801080===

Revision as of 18:09, 26 September 2012

Expression of Thaumatin

Ion exchange chromatography

Experimental Results

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.

Experimental details:

  • Samples: cell lysate, supernatant from culture, reference for thaumatin (MedHerbs)
  • Dialysis against 20mM MES Buffer pH 6.0 (twice) using a 12-16 kDa dialysis membrane
  • Chromatography with a Äkta purifier equipped with a Ressource S 6ml (S: Methyl sulfonate (strong cation exchanger))
  • Sample was applied using a super-loop
  • Wash with two column volumes 20mM MES Buffer pH 6.0
  • Elution with a gradient of 0 - 500 mM NaCl over 5 column volumes
  • Fractions of 1 ml were collected during the elution

Experimental results:

  • Reference Thaumatin (see figure A and B)
    • nearly no protein in the flow through during sample application (see figure A)
    • a major peak eluting at ~14 mS*cm-1 which corresponds to fraction Nr. 14 see figure A)
    • The corresponding SDS-PAGE showed a clear band around fraction Nr. 14 corresponding matching the expected 22 kDa
  • Cell lysate
    • high concentration of protein in the flow through (see figure C and D)
    • no clear peak around fraction 14 could be detected
    • 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.
  • Supernatant of yeast culture (see figure E and F)
    • Export of the protein in high concentrations was unlikely to happen, therefore 48 ml of supernatant were loaded on the column
    • Ratio between flowthrough and eluted protein was less favorable compared to the cell lysate.
    • Total protein quantities were to low to be detected by Comassie stain, therefore a silver stain was performed (see figure F)
    • Beside the reference no additional proteinbands could be detected on the silver stained SDS-PAGE

Conclusion from this experiment:
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.

Applications of BBa_K801080

User Reviews

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