Methods for preparation of active separase
AssigneeThe Regents of the University of California
InventorsLaura E. Rosen, David O. Morgan
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Abstract
Provided herein are polypeptide constructs containing a securin, e.g., a full-length securin or a truncated securin, fused to a separase. The polypeptide constructs may further contain linker peptides, protease recognition sites, and unfoldase recognition sites to facilitate expression and/or purification. Methods for obtaining the polypeptide constructs with active separase activity are also described, as well as methods for identify separase modulator compounds such as separase inhibitors.
Claims33
A polypeptide construct comprising a securin fused to a separase.
A polypeptide construct comprising a securin linked to an unfoldase recognition site.
The mixture of claim 29, wherein the test substance is an organic small-molecule separase inhibitor candidate.
A method for identifying a separase modulator compound, the method comprising:
- (i) measuring a level or rate of peptide substrate cleavage by a polypeptide construct in the presence of a candidate compound, wherein the polypeptide construct comprises a securin fused to a separase;
- (ii) measuring a level or rate of peptide substrate cleavage by the polypeptide construct in the absence of the candidate compound;
- and
- (iii) identifying the candidate compound as a separase modulator compound when the level or rate of peptide substrate cleavage in step (i) is higher or lower than the level or rate of peptide substrate cleavage in step (ii).
A method for obtaining an active separase fusion protein, the method comprising expressing a polypeptide comprising a truncated securin fused to a separase, thereby obtaining the active separase fusion protein.
A method for obtaining an active separase, the method comprising:
- (a) co-expressing a separase and a securin, wherein the securin is linked to an unfoldase recognition site;
- and
- (b) combining the co-expressed separase and securin with an unfoldase-peptidase complex;
- thereby removing the securin and obtaining the active separase.
A method for obtaining an active separase, the method comprising:
- (1) expressing a polypeptide comprising a securin fused to a separase;
- and
- (2) removing the securin from the expressed polypeptide, thereby obtaining the active separase;
- wherein the active separase is substantially free of the securin.
An isolated active separase, which is substantially free of securin.
Dependencies are inferred from the claim text.