High throughput screen for biologically active polypeptides
Methods for screening libraries of polypeptides for biologically activity on cells. For example, polypeptides can be synthesized and encapsulated along with their coding sequences in microcapsules of an emulsion. Emulsion microcapsules can then be fused with microcapsules comprising test cells and biological activity on the cells is assessed to identify biologically active polypeptides and nucleic acid molecules encoding the same.
1. A method of providing a nucleic acid molecule encoding an active polypeptide having a desired activity, the method comprising the steps of:
(a) obtaining a population of nucleic acid molecules comprising sequences that encode polypeptides, wherein individual members of the population encode different polypeptides and are bound to a carrier, to provide a population of carrier-bound nucleic acid molecules;
(b) encapsulating individual carrier-bound nucleic acid molecules in microcapsules and incubating the encapsulated carrier-bound nucleic acid molecules under conditions that permit expression of polypeptides, such that the encoded polypeptide molecule becomes selectively associated on said carrier with at least one copy of the nucleic acid molecule that encodes it within its respective microcapsule;
(c) breaking the microcapsules and dissociating individual polypeptides from their respective carriers and the nucleic acid that encodes it and testing the individual dissociated polypeptides for a response to a target molecule; and
(d) identifying polypeptides that provide a response to the target molecules, to provide the nucleic acid molecule encoding the active polypeptide.
2. The method of claim 1 , wherein the target molecule comprises a test cell.
3. The method of claim 2 , wherein the target molecule is expressed in the surface of the test cell.
4. The method of claim 2 , wherein (c) testing a target molecule for a response to individual member polypeptides of the population comprises testing cells for a biological response to individual member polypeptides of the population.
5. The method of claim 2 , wherein the cells are bacterial cells, fungal cells, insect cells or mammalian cells.
6. The method of claim 2 , wherein testing cells for a biological response comprises detecting a change in the optical or fluorescent properties of the cells.
7. The method of claim 6 , wherein detecting a change in the optical or fluorescent properties of the cells comprises detecting binding of a labeled reagent to the cells.
8. The method of claim 7 , wherein the labeled reagent comprises a fluorescent label.
9. The method of claim 7 , wherein the labeled reagent is an antibody.
10. The method of claim 2 , wherein testing the cells for a biological response to the polypeptide comprises determining whether the nucleic acid molecules are bound to the component of the test cells.
11. The method of claim 1 , wherein testing a target molecule for a response to individual member polypeptides of the population comprises contacting the member polypeptides with target molecules isolated in a gel, a well of micro titer plate or a microcapsule of an emulsion.
12. The method of claim 1 , wherein each polypeptide molecule is associated with at least one copy of the nucleic acid molecule that encodes it in a gel, a well of micro titer plate or a microcapsule of an emulsion.
13. The method of claim 1 , wherein identifying the sequence of nucleic acid molecules associated with polypeptides comprises sequencing the nucleic acid molecules.
14. The method of claim 1 , wherein the population of nucleic acid molecules comprises sequences that encode antibody polypeptides.
15. The method of claim 1 , wherein the polypeptide molecules are dissociated from the at least one copy of the nucleic acid molecule that encodes it prior to said testing.
16. The method of claim 15 , further comprising contacting the polypeptide molecules with a dissociation agent prior to said testing.
17. The method of claim 1 , wherein the response to individual member polypeptides of the population comprises binding of the individual member polypeptides to the target molecule.
18. The method of claim 1 , wherein the nucleic acid molecules are bound to a detectable tag.
19. The method of claim 1 , wherein the carrier is a carrier bead.
20. The method of claim 19 , wherein the nucleic acid molecules and/or carrier bead comprise a detectable label.
21. The method according to claim 1 ,
wherein the polypeptides are antibody polypeptides.