Methods for preparing purified prostaglandin E synthase
OwnerPer-Johan Jakobsson, Rolf Weinander +2inventors
InventorsPer-Johan Jakobsson, Rolf Weinander, Ralf Morgenstern, Ralph H. Lambalot
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Abstract
The invention provides purified preparations of prostaglandin E synthase (PGES), analogs and variants thereof, such as portions thereof, and fusion polypeptides comprising these. The invention also provides methods for obtaining these purified preparations; methods for identifying modulators of PGES, e.g., small molecules inhibiting or stimulating the activity of PGES; and methods for preparing prostaglandins. Nucleic acids encoding PGES polypeptides of the invention are also provided.
Claims55
A substantially pure preparation of Prostaglandin E Synthase (PGES) comprising SEQ ID NO 2, or analog or variant thereof.
A method for obtaining a substantially pure preparation of a PGES fusion polypeptide, comprising
- (i) providing a lysate comprising a PGES fusion polypeptide comprising a PGES polypeptide, analog or fragment thereof fused to a heterologous polypeptide capable of binding to a metal ion affinity chromatography;
- (ii) subjecting the lysate of (i) to hydroxyapatite (HA) chromatography;
- (iii) obtaining the unbound fraction from the HA chromatography;
- (iv) subjecting the unbound fraction from the HA chromatography to metal ion affinity chromatography, in conditions appropriate for the fusion polypetide to bind to the metal ion;
- and
- (v) eluting the metal ion affiniyt chromatography, to thereby obtain a substantially pure preparation of a PGES fusion polypeptide.
A method for identifying an agent which modulates the interaction between a PGES polypeptide or analog or fragment thereof and a PGES-binding partner, comprising contacting a substantially pure PGES polypeptide or analog or fragment thereof;
- a PGES-binding partner;
- and a test agent, in conditions under which, but for the presence of the test agent, the PGES polypeptide or analog or fragment thereof and the PGES binding partner interact, wherein a difference in the interaction between the PGES polypeptide or analog or fragment thereof and the PGES-binding partner indicates that the test agent modulates their interaction.
A method for treating a subject in need of modulation of PGES or prostaglandin activities, comprising administering to the subject a pharmaceutically efficient amount of a modulator of PGES, such that the subject is treated.
An isolated nucleic acid encoding a human PGES polypeptide comprising the amino acid sequence set forth in SEQ ID NO:
- 2 or which differs from the sequence in 1 to about 20 amino acids, which PGES polypeptide is fused to a His6 tag.
Dependencies are inferred from the claim text.