Expression and purification of human PYY(3-36) in Escherichia coli using a His-tagged small ubiquitin-like modifier fusion

Christopher H. Fazen, Anna R. Kahkoska, Robert Patrick Doyle

Research output: Contribution to journalArticle

5 Citations (Scopus)

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 (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.

Original languageEnglish (US)
Pages (from-to)51-59
Number of pages9
JournalProtein Expression and Purification
Volume85
Issue number1
DOIs
StatePublished - Sep 2012

Fingerprint

Ubiquitin
Peptide Hydrolases
Escherichia coli
Digestion
Factor Xa
Affinity Chromatography
Metals
Appetite Regulation
Antibodies
Reverse-Phase Chromatography
Circular Dichroism
Mass Spectrometry
Spectrum Analysis
Western Blotting
High Pressure Liquid Chromatography
Hormones
Amino Acids
Proteins

Keywords

  • E. coli
  • Factor Xa
  • HPLC
  • IMAC
  • PYY
  • SUMO

ASJC Scopus subject areas

  • Biotechnology

Cite this

Expression and purification of human PYY(3-36) in Escherichia coli using a His-tagged small ubiquitin-like modifier fusion. / Fazen, Christopher H.; Kahkoska, Anna R.; Doyle, Robert Patrick.

In: Protein Expression and Purification, Vol. 85, No. 1, 09.2012, p. 51-59.

Research output: Contribution to journalArticle

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