Protein discovery using intracellular ribosome display

1. A method comprising:

providing a construct comprising a deoxyribonucleic acid molecule encoding a protein which binds to a target molecule, said deoxyribonucleic acid molecule being coupled to a stall sequence;

transforming a host cell with the construct;

culturing the host cell under conditions effective to form a complex within the host cell, the complex comprising the protein whose translation has been stalled, an mRNA encoding the protein, and ribosomes, wherein the protein in the complex is in a properly folded, active form;

recovering the complex from the cell; and

isolating the mRNA from the recovered complex.

2. The method of claim 1 further comprising:

reverse transcribing the isolated mRNA to form a cDNA encoding the protein;

forming a construct comprising the cDNA coupled to the stall sequence; and

repeating said transforming, said culturing, and said recovering to enrich the protein recovered.

3. The method of claim 1 , wherein said isolating comprises:

dissociating the complex.

4. The method of claim 3 , wherein said dissociating is carried out with EDTA.

5. The method of claim 2 further comprising:

characterizing enrichment of the protein by sequencing or ELISA.

6. The method of claim 2 , wherein said isolating, said reverse transcribing, said forming, and said repeating are carried out multiple times.

7. The method of claim 1 , wherein the stall sequence is SecM coupled to the deoxyribonucleic acid molecule.

8. The method of claim 7 , wherein the construct further comprises:

an epitope flag,

a c-Myc epitope tag,

a 6×-His tag,

a thrombin cleavage site,

a linker, and

a stop codon; wherein the c-Myc epitope tag, the 6×-His tag, the thrombin cleavage site, and the linker are all positioned within the construct between the deoxyribonucleic acid molecule and the SecM stalling sequence.

9. The method of claim 1 , wherein said recovering is carried out by affinity selection with an agent specific for the protein.

10. The method of claim 1 , wherein the protein is a single-chain variable fragment antibody.

11. The method of claim 1 , wherein the cell is a bacterial cell.

12. The method of claim 11 , wherein the bacterial cell is E. coli.

13. The method of claim 1 , wherein the deoxyribonucleic acid molecule is coupled to the stall sequence by way of a linker sequence.

14. The method of claim 13 , wherein the linker sequence is selected from the group consisting of (i) a Glycine-Serine linker comprising 10 to 50 Glycine/Serine units and (ii) a linker sequence having the amino acid sequence of SEQ ID NO:27.

15. The method of claim 2 further comprising: characterizing the enriched protein.

16. The method of claim 9 , wherein the agent is the target molecule.

17. The method of claim 12 , wherein the bacterial cell is an Origami strain of E. coli.

18. The method of claim 1 , wherein the construct comprises two or more deoxyribonucleic acid molecules encoding two or more proteins which bind to the target molecule.

19. A method comprising:

providing a construct comprising a deoxyribonucleic acid molecule encoding a protein which binds to a target molecule, said deoxyribonucleic acid molecule being coupled to a stall sequence;

transforming a host cell with the construct; and

culturing the host cell under conditions effective to form a complex within the host cell, the complex comprising the protein whose translation has been stalled, an mRNA encoding the protein, and ribosomes, wherein the protein in the complex is in a properly folded, active form, wherein the protein is a ligand binding protein selected from the group consisting of high-affinity antibody fragments, single-chain Fv antibody fragments, nanobodies, nanobody fragments, fluorobodies, and aptamers.

20. The method of claim 19 further comprising:

recovering the complex from the cell;

isolating the mRNA from the recovered complex;

reverse transcribing the isolated mRNA to form a cDNA encoding the protein;

forming a construct comprising the cDNA coupled to the stall sequence; and

repeating said transforming, said culturing, and said recovering to enrich the protein recovered.

21. The method of claim 20 , wherein said isolating comprises:

dissociating the complex.

22. The method of claim 20 further comprising: characterizing the enriched ligand binding protein.

23. The method of claim 20 , wherein said isolating, said reverse transcribing, said forming, and said repeating are carried out multiple times.

24. The method of claim 19 , wherein the stall sequence is SecM coupled to the deoxyribonucleic acid molecule.

25. The method of claim 19 , wherein said recovering is carried out by affinity selection with the target molecule.

26. The method of claim 19 , wherein the protein is a single-chain variable fragment antibody.

27. The method of claim 19 , wherein the cell is a bacterial cell.

28. The method of claim 27 , wherein the bacterial cell is E. coli.

29. The method of claim 28 , wherein the bacterial cell is an Origami strain of E. coli.

30. The method of claim 19 , wherein the deoxyribonucleic acid molecule is coupled to the stall sequence by way of a linker sequence.

31. The method of claim 30 , wherein the linker sequence is selected from the group consisting of (i) a Glycine-Serine linker comprising 10 to 50 Glycine/Serine units and (ii) a linker sequence having the amino acid sequence of SEQ ID NO:27.

32. The method of claim 19 , wherein the construct comprises two or more deoxyribonucleic acid molecules encoding two or more proteins which bind to the target molecule.

33. The method of claim 1 , wherein the stall sequence has the amino acid sequence of FXXXXWIXXXXGIRAGP (SEQ ID NO: 26), wherein X is any amino acid.

34. The method of claim 19 , wherein the stall sequence has the amino acid sequence of FXXXXWIXXXXGIRAGP (SEQ ID NO: 26), wherein X is any amino acid.