Reverse n-hybrid screening method
OwnerRichard Hopkins, Ilya Serebriiskii +2inventors
InventorsRichard Hopkins, Ilya Serebriiskii, Paul Michael Watt, Erica Golemis
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Abstract
The present invention relates provides an improved reverse n-hybrid screening method for identifying antagonists or inhibitors of biological interactions, wherein multiple reporter genes are employed to distinguish antagonists or inhibitors of one interaction from those antagonists or inhibitors of other interactions, particularly where each of said interactions involves one or more common interacting partners. A further aspect of the invention provides novel expression vectors for performing the inventive method, particularly in high throughput screens.
Claims55
A method of identifying a peptide that partially or completely inhibits a target interaction between two or more binding partners in a host cell but does not inhibit a non-target interaction between some but not all of said binding partners, said method comprising:
- (i) expressing in a cellular host:
- (a) the binding partners of said target interaction such that they operably control the expression of one or more reporter genes in said cellular host, wherein said expression is partially or completely inhibited by disruption of said target interaction;
- (b) the binding partners of said non-target interaction such that they operably control the expression of one or more reporter genes in said cellular host, wherein said expression is partially or completely inhibited by disruption of said non-target interaction and wherein said reporter gene is distinct from the reporter gene(s) expressed under control of the target interaction;
- and (c) a candidate peptide;
- (ii) growing the cellular host under conditions sufficient to distinguish the expression of each reporter gene(s) at (a) from expression of the reporter gene(s) at (b);
- and
- (iii) detecting those host cells wherein expression of the reporter gene(s) operably under control of the target interaction is(are) partially or completely inhibited and expression of the reporter gene(s) operably under control of the non-target interaction is(are) not inhibited, said detected cells expressing a peptide that partially or completely inhibits the target interaction.
A method of identifying a peptide that partially or completely inhibits a target interaction between two or more binding partners in a yeast cell but does not inhibit a non-target interaction between some but not all of said binding partners, said method comprising:
- (i) transforming a yeast cell with a vector selected from the group consisting of:
- pBLOCK-3.0 (SEQ ID NO:
- 1);
- pBLOCK-3.2 (SEQ ID NO:
- 2);
- pBLOCK-3.4 (SEQ ID NO:
- 3);
- pBLOCK-3.6 (SEQ ID NO:
- 4);
- pBLOCK-3.8 (SEQ ID NO:
- 5);
- pBLOCK-3.9 (SEQ ID NO:
- 6);
- pBLOCK-3.10 (SEQ ID NO:
- 7);
- pBLOCK-3.11 (SEQ ID NO:
- 8);
- and pBLOCK-4.0 (SEQ ID NO:
- 9), wherein said vector further comprises nucleic acid encoding a candidate peptide being tested for inhibitory activity;
- (ii) introducing to said transformed yeast cell nucleic acid encoding:
- (a) the binding partners of said target interaction such that they operably control the expression of one or more counter selectable reporter genes or fluorescent protein-encoding reporter genes in said cellular host, wherein said expression is partially or completely inhibited by disruption of said target interaction;
- and (b) the binding partners of said non-target interaction such that they operably control the expression of one or more counter selectable reporter genes or fluorescent protein-encoding reporter genes in said cellular host, wherein said expression is partially or completely inhibited by disruption of said non-target interaction and wherein said reporter gene is distinct from the reporter gene(s) expressed under control of the target interaction;
- (iii) selecting the recombinants;
- (iv) growing the recombinants under conditions sufficient to distinguish the expression of each reporter gene(s) at (a) from expression of the reporter gene(s) at (b);
- and
- (v) detecting those host cells wherein expression of the reporter gene(s) operably under control of the target interaction is(are) partially or completely inhibited and expression of the reporter gene(s) operably under control of the non-target interaction is(are) not inhibited, said detected cells expressing a peptide that partially or completely inhibits the target interaction.
A yeast shuttle vector comprising a nucleotide sequence substantially as set forth in a sequence selected from the group consisting of SEQ ID NOs:
- 1 to 10 and a functionally equivalent variant or derivative of any one of said sequence.
A vector for expressing red and green fluorescent proteins in yeast comprising:
- (i) a green fluorescent protein expression cassette comprising the gfp gene operably under control of a chimeric yeast operable LexA/GAL1 promoter having multiple LexA operator sites;
- and
- (ii) a red fluorescent protein expression cassette comprising the cobA gene operably under control of a chimeric cl/GAL1 promoter having multiple cl operator sites.
Dependencies are inferred from the claim text.