Affinity purification of recombinant protein-tyrosine phosphatase 1B using a highly selective inhibitor.
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.
Pedersen
Anja K
AK
Department of Medicinal Chemistry, Danish University of Pharmaceutical Sciences, DK-2100 Copenhagen, Denmark.
Branner
Sven
S
Mortensen
Steen B
SB
Andersen
H Sune
HS
Klausen
Kirsten M
KM
Møller
Karin B
KB
Møller
Niels Peter H
NP
Iversen
Lars F
LF
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
D018076:DNA, Complementary; D004791:Enzyme Inhibitors; D011994:Recombinant Proteins; D054562:Protein Tyrosine Phosphatase, Non-Receptor Type 1; D017027:Protein Tyrosine Phosphatases