| Article ID | Journal | Published Year | Pages | File Type |
|---|---|---|---|---|
| 2020673 | Protein Expression and Purification | 2012 | 9 Pages |
Human PYY(3–36) (hPYY3–36) is a 34 amino acid hormone that has received a great deal of attention due to its effects on appetite regulation. hPYY(3–36) was modified at the N-terminus with an octahistidine tag and factor Xa protease sequence along with the small ubiquitin-like modifier (SUMO) tag and expressed in Escherichia coli. The protein was purified from clarified E. coli lysate by immobilized metal affinity chromatography (IMAC) with a yield of 30 ± 7 mg/L of induced culture returned as an average over seven runs, and its identity was confirmed by Western blot and hPYY antibody recognition. The SUMO-tagged hPYY(3–36) was digested with two different proteases to return either His-tagged hPYY(3–36) or unmodified hPYY(3–36): (1) digestion with SUMO protease proceeded at about 50% efficiency yielding His-tagged hPYY(3–36); (2) digestion with factor Xa protease proceeded at greater than 90% efficiency yielding final hPYY(3–36). Products were purified from the digestion mixtures by reverse-phase high-performance liquid chromatography (C18) or IMAC, respectively, the identities were confirmed by mass spectrometry and hPYY antibody recognition, and the folded state of His-tagged hPYY(3–36) was investigated by circular dichroism spectroscopy.
► The recombinant expression of PYY3–36 in Escherichia coli is described. ► The small ubiquitin like modifier (SUMO) system allowed good yields >30 mg/L to be isolated and purified. ► Purification was through IMAC to produce HIS-tagged PYY3–36 or HPLC to produce un-tagged peptide. ► Peptide was identified and characterized by SDS–PAGE, Western blot, ELISA, CD and MALDI-Tof MS.
