Affinity purification of recombinant protein-tyrosine phosphatase 1B using a highly selective inhibitor |
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Authors: | Pedersen Anja K Branner Sven Mortensen Steen B Andersen H Sune Klausen Kirsten M Møller Karin B Møller Niels Peter H Iversen Lars F |
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Institution: | Department of Medicinal Chemistry, Danish University of Pharmaceutical Sciences, DK-2100 Copenhagen, Denmark. |
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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-thieno2,3-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. |
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Keywords: | Affinity purification Inhibitor Crystallization Protein-tyrosine phosphatases |
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