title: Affinity purification of recombinant protein-tyrosine phosphatase 1B using a highly selective inhibitor.
abstract: Our structure-based drug discovery program within the field of protein-tyrosine phosphatases (PTPs) demands delivery of significant amounts of protein with extraordinary purity specifications over prolonged time periods. Hence, replacement of classical, multi-step, low-yield protein purifications with efficient affinity techniques would be desirable. For this purpose, the highly selective PTP1B inhibitor 2-(oxalyl-amino)-4,5,6,7-tetrahydro-thieno[2,3-c]pyridine-3-carboxylic acid (OTP) was coupled to epoxy-activated Sepharose 6B (OTP Sepharose) and used for one-step affinity purification of tag-free PTP1B. The elution was performed with a combined pH and salt gradient. Importantly, since OTP Sepharose binds PTP1B with an intact active site only, the method ensures that the purified enzyme is fully active, a feature that might be particularly important in PTP research.
authors.affiliation: Department of Medicinal Chemistry, Danish University of Pharmaceutical Sciences, DK-2100 Copenhagen, Denmark.
authors.forename: Anja K
authors.initials: AK
authors.lastname: Pedersen
authors.forename: Sven
authors.initials: S
authors.lastname: Branner
authors.forename: Steen B
authors.initials: SB
authors.lastname: Mortensen
authors.forename: H Sune
authors.initials: HS
authors.lastname: Andersen
authors.forename: Kirsten M
authors.initials: KM
authors.lastname: Klausen
authors.forename: Karin B
authors.initials: KB
authors.lastname: Møller
authors.forename: Niels Peter H
authors.initials: NP
authors.lastname: Møller
authors.forename: Lars F
authors.initials: LF
authors.lastname: Iversen
mesh_terms: D020134:Catalytic Domain; D002846:Chromatography, Affinity / Q000379:methods*; D002850:Chromatography, Gel; D003001:Cloning, Molecular; D018076:DNA, Complementary; D004791:Enzyme Inhibitors / Q000494:pharmacology*; D006863:Hydrogen-Ion Concentration; D054562:Protein Tyrosine Phosphatase, Non-Receptor Type 1; D017027:Protein Tyrosine Phosphatases / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification*; D011994:Recombinant Proteins / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification
chemical_list: D018076:DNA, Complementary; D004791:Enzyme Inhibitors; D011994:Recombinant Proteins; D054562:Protein Tyrosine Phosphatase, Non-Receptor Type 1; D017027:Protein Tyrosine Phosphatases