CPC G01N 33/6845 (2013.01) [C07K 16/18 (2013.01); C40B 30/04 (2013.01); G01N 33/5008 (2013.01); G01N 33/533 (2013.01); G01N 33/542 (2013.01); C07K 2319/70 (2013.01)] | 2 Claims |
1. A method for screening a compound capable of modulating the binding between a polypeptide (A) and a polypeptide (B) comprising the step of
i) providing a cell that expresses:
(a) a polypeptide (GFP1-9) comprising an amino acid sequence having at least 90% of identity with the amino acid sequence selected from the group consisting of SEQ ID NO:1-4
(b) a first fusion protein wherein the polypeptide (A) is fused to a polypeptide (GFP10) having an amino an amino acid sequence having at least 90% of identity with the amino acid sequence selected from the group consisting of SEQ ID NO:5-7
(c) a second fusion protein wherein the polypeptide (B) is fused to a polypeptide (GFP11) having an amino an amino acid sequence having at least 90% of identity with the amino acid sequence selected from the group consisting of SEQ ID NO:8-9
and (d) an intrabody specific for the complex formed by the self-assembly of the first, second and third polypeptides (a), (b) and (c), wherein the intrabody is a single domain antibody comprising the three complementarity determining regions (CDRs) of SEQ ID NO: 16;
ii) contacting the cell with a candidate compound,
iii) detecting the fluorescence; and
iv) positively selecting the candidate compound when the fluorescence is modulated compared to fluorescence detected in the absence of the candidate compound,
wherein the polypeptide (A) is a GTPase and the polypeptide (B) is a GTPase binding domain (GBD) or vice versa.
|