Methods and means for screening for rhomboid activity
AssigneeMedical Research Council
InventorKeith H. Ansell
Life of this patent
Estimating the term…
Abstract
This invention relates to methods of screening for rhomboid modulating compounds using a substrate polypeptide has a core domain comprising a rhomboid cleavable TMD sequence linked to an upstream tag sequence. The core domain sequence is not susceptible to cleavage by non-rhomboid proteases so products of rhomboid dependent proteolysis products may be detected by determining the presence of the tag sequence. Rhomboid modulating compounds identified by the present methods may be useful in a range of therapeutic applications.
Claims49
A method for identifying and/or obtaining a modulator of a rhomboid polypeptiide, which method comprises:
- (a) contacting a rhomboid polypeptide and a substrate polypeptide in the presence of a test compound and one or more non-rhomboid proteases,
- wherein said substrate polypeptide comprises a core domain which has a rhomboid cleavable TMD sequence linked to an upstream tag sequence, the core domain sequence not being susceptible to cleavage by the one or more non-rhomboid proteases, and;
- (b,) determining the presence or amount in said medium of a soluble polypeptide fragment comprising said tag sequence.
A polypeptide which is proteolytically cleavable by a Rhomboid polypeptide, said polypeptide comprising an a core domain which has a rhomboid cleavable TMD sequence linked to an upstream tag sequence, the core domain sequence not being susceptible to cleavage by mammalian metalloproteases.
A method for obtaining a cleavage product of a Rhomboid polypeptide, which method comprises:
- (a) contacting a Rhomboid polypeptide and a substrate polypeptide and one or more non-rhomboid proteases, wherein said substrate polypeptide comprises a core domain which has a rhomboid cleavable TMD sequence linked to an upstream tag sequence, the core domain sequence not being susceptible to cleavage by the one or more non-rhomboid proteases, and;
- (b) contacting said medium with an antibody which binds to said tag sequence, and
- (c) isolating/purifying soluble polypeptide fragment bound to said antibody.
Dependencies are inferred from the claim text.