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| Code | CSB-EP001899HU |
| MSDS | |
| Size | US$306 |
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CUSABIO synthesized the recombinant gene by integrating the N-terminal 6xHis tag sequence into the targeted gene encoding the 1-732aa of the human APEH. The synthesized gene was subsequently cloned into an expression vector. After cloning, the expression vector was introduced into the E.coli for expression. The product was purified to obtain the recombinant human APEH protein carrying N-terminal 6xHis tag. The SDS-PAGE assayed the purity of this recombinant APEH protein greater than 85%. This APEH protein migrated along the gel to a band of about 90 kDa molecular weight.
APEH is a gene providing instruction of making a protein called acylamino-acid-releasing enzyme in human and belongs to peptidase S9C family. This protein, also abbreviated APH or AARE, is a tetrameric serine protease which specifically removes acetyl amino acids from N-terminally acetylated peptides and play an important role in destroying oxidatively-damaged proteins in living cells. APH preferentially cleaves off Ac-Ala, Ac-Met and Ac-Ser. Deletions of this gene locus are found in various types of carcinomas, including small-cell lung carcinoma and renal cell carcinoma.
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We have been trying to validate its activity using AANA as a substrate (Nitroanaline yellow product produced and followed at 405 nm) without luck. I'm hoping you can give us advice.
We dissolved the protein in 100 uL of DI water (1ug/ul). We used 1, 2, and 4 ug protein in 10 mM TRIS (pH = 8.0) with 5 mM AANA (total volume 200 uL), incubated at 37C. We haven't seen any yellow color develop but we do with our Hela cell lysate controls. We've published this before: https://www.sciencedirect.com/science/article/pii/S0009279721002775
Please let me know if you have suggestions on how we can test the activity of this protein--we want to use it in a kinetic assay.
The protein (CSB-EP001899HU) is obtained by renaturation of the inclusion body. Although renaturation reaches a soluble buffer (without denaturing agent), there is no guarantee that the protein after renaturation must be active;
You can try to use APEH protein and LGALS8 protein (CSB-EP012894HU) for functional Elisa verification to see if the protein is active;
Reference link: https://www.uniprot.org/uniprotkb/P13798/entry#interaction
If the above two proteins do not interact, we will try to optimize the expression of CSB-EP001899HU supernatant or you can consider buying eukaryotic expression system proteins such as Yeast and Mammalian Cells.
