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

Revision as of 18:20, 26 September 2012

Expression of Thaumatin

Characterization of this BioBrick was carried out using the yeast expression vector BBa_K801004 for transient transfection with the yeast strain INVSc1.

Ion exchange chromatography

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

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