Part:BBa_K3105672
HRP-2A-AAO
Part to co-express horseradish peroxidase (HRP) and aryl-alcohol oxidase (AAO) in Pichia pastoris. The 2A-peptide will facilitate cleavage of the polypeptide during translation, leading to separate units of HRP and AAO.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21INCOMPATIBLE WITH RFC[21]Illegal BglII site found at 880
Illegal BamHI site found at 1275
Illegal BamHI site found at 1462 - 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal AgeI site found at 961
Illegal AgeI site found at 2422 - 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. patoris 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 different assays. For HRP activity, an ABTS-assay was used and the activity between uninduced and induced samples was compared (Figure 2A). Clear increase
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