Patent Number: 6,165,731

Title: Assay for the ubiquitination-promoting activity of human proteins

Abstract: A method is provided for identifying an compound that affects an activity of a polypeptide subunit of a SCF complex. The method includes contacting a sample comprising a chimeric SCF complex assembled from subunits derived from Saccharomyces cerevisiae or human and another species and a CDC34p polypeptide with the compound under conditions that allow the components to interact, and adding to these components an E1 enzyme, ubiquitin and ATP, and a SCF substrate. The ubiquitination of the SCF substrate is measured. A chimeric in vitro assay system is provided for measuring CDC53p or CUL1p activity, comprising a CDC4p, CDC34p, and a SKP1p polypeptide, and either a CDC53p or CUL1p polypeptide. In this assay the CDC4p, CDC34p, and SKP1p polypeptide are either a yeast polypeptide or a polypeptide from another species, and at least one of the CDC4p, CDC34p, and SKP1p polypeptides is a yeast polypeptide and at least one of the CDC4p, CDC34p, and SKP1p polypeptides is a polypeptide from another species. A method is further provided for identifying a compound that affects the ability of a CDC4p, a SKP1p, a CDC34p, and a CDC53p or a CUL1p to ubiquitinate a substrate. The method includes contacting a sample comprising a CDC4p, a SKP1p, a CDC34p, and a CDC53p or CUL1p, with the compound under conditions sufficient to allow the components to interact, and adding to these components an E1 enzyme, ubiquitin and ATP, and a substrate for ubiquitination. The ability of the CDC4p, the SKP1p, the CDC34p, and the CDC53p or CUL1p, to ubiquitinate the substrate is measured. A method is also provided of identifying a polypeptide having a function of a CDC4 subunit of SCF. A method is further provided for identifying a polypeptide as a substrate for a ubiquitination reaction.

Inventors: Deshaies; Raymond (Claremont, CA), Lyapina; Svetlana (South Pasadena, CA), Correll; Craig C. (Pasadena, CA)

Assignee: California Institute of Technology

International Classification: C07K 16/14 (20060101); C07K 16/18 (20060101); C12Q 1/25 (20060101); G01N 033/53 (); G01N 033/573 (); C12P 013/18 (); A61K 039/395 (); C07K 016/00 ()

Expiration Date: 12/26/2013