title: Expression and purification of human PYY(3-36) in Escherichia coli using a His-tagged small ubiquitin-like modifier fusion.
abstract: 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 (C(18)) 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.
authors: Department of Chemistry, Center for Science and Technology, Syracuse University, Syracuse, NY 13244, USA.
authors: Christopher H
authors: CH
authors: Fazen
authors: Anna R
authors: AR
authors: Kahkoska
authors: Robert P
authors: RP
authors: Doyle
mesh_terms: D000595:Amino Acid Sequence; D002846:Chromatography, Affinity; D002851:Chromatography, High Pressure Liquid; D002942:Circular Dichroism; D003001:Cloning, Molecular / Q000379:methods*; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000235:genetics*; D006639:Histidine / Q000737:chemistry; D006801:Humans; D008969:Molecular Sequence Data; D019894:Peptide YY / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification; D011487:Protein Conformation; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification; D025841:Small Ubiquitin-Related Modifier Proteins / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
chemical_list: D011993:Recombinant Fusion Proteins; D025841:Small Ubiquitin-Related Modifier Proteins; D019894:Peptide YY; D006639:Histidine