Part:BBa_K3105679
Expression construct HRP-2A-AAO
Expression circuit for HRP and AAO in Pichia pastoris. HRP will be secreted due to the addition of the α-factor secretion signal and AAO will remain in the cell. The 2A-selfcleaving peptide will lead to cleavage of the polypeptide, achieving separation of HRP from AAO.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21INCOMPATIBLE WITH RFC[21]Illegal BglII site found at 2086
Illegal BamHI site found at 2481
Illegal BamHI site found at 2668
Illegal XhoI site found at 1183 - 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal AgeI site found at 2167
Illegal AgeI site found at 3628
Illegal AgeI site found at 4241 - 1000COMPATIBLE WITH RFC[1000]
Results
The part was expressed in X-33 P. pastoris cells using the pPICZαB shuttle vector. Cultures were induced with methanol and analysed on SDS-PAGE. HRP and AAO was successfully expressed (Figure 1). Furthermore, also the functionality of the 2A-peptide in P. pastoris was shown, because failure in cleavage would lead to a induction band bigger than 100 kDa.
X-33 P. pastoris cells were transformed with pPICZαB_HRP-2A-AAO and expression cultures were induced. Different fractions (pellet (P) and supernatant (S) samples / uninduced (u) and induced (i) cultures) from X-33 HRP-2A-AAO expression culture were analysed on a 10 % SDS-PAGE stained with Coomassie Blue. After 24 h an induction band can be seen at around 55 kDa, which is approximately the calculated molecular weight for both HRP and AAO. This shows that the enzymes are expressed as well as that the cleavage initiated by the 2A-peptide is functional.
Enzyme activity was assessed by conducting colorimetric assays. For HRP activity, ABTS was used as oxidized substrate, which leads to higher Absorbance at 405 nm. Activity between uninduced and induced samples was compared (Figure 2A). A clear increase in absorbance in induced samples, while uninduced samples remain at a value of 0, proves enzyme activity of HRP.
To test the activity of AAO, FOX-assay was used, which is also based on a increase of absorbance (560 nm). Comparing absorbance values of uninduced and induced samples show a significant increase in the induced samples (Figure 2B), indicating the functional expression of AAO. Thus, the induction band in Figure 1 contains both enzymes.
The activities of the two enzymes expressed in Figure 5 were assessed using two different enzymatic assays. Uninduced, orange; induced, green. Assays were performed in triplicates.
(A) ABTS-assay for HRP. (B) FOX-assay for AAO activity. Two different volumes of lysate were used and absorbance at 560 nm was measured. The difference in activity between uninduced and induced samples was statistically significant as measured by paired t-test (*= p<0.05 and ****= p<0.00001, respectively).
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