Protein screening methods
The invention provides methods and compositions useful for identifying polypeptides with desired characteristics in vitro.
1. An X-display complex comprising:
(a) a nucleic acid molecule comprising a polypeptide-encoding sequence, wherein the nucleic acid molecule is covalently bound to a first high affinity ligand;
(b) a polypeptide encoded by the nucleic acid molecule, wherein the polypeptide is covalently linked at the C-terminal residue through a peptide acceptor to a second high affinity ligand, and wherein the peptide acceptor is selected from the group consisting of puromycin, pyrazolopyrimidine, phenylalanyl-adenosine, tyrosyl adenosine, alanyl adenosine, phenylalanyl 3′ deoxy 3′ amino adenosine, alanyl 3′ deoxy 3′ amino adenosine, and tyrosyl 3′ deoxy 3′ amino adenosine; and
(c) a single multimeric ligand acceptor protein, wherein the single multimeric ligand acceptor protein is bound noncovalently and directly to both the first and second high affinity ligand at distinct ligand binding sites on the ligand acceptor protein, thereby noncovalently linking the nucleic acid molecule and the polypeptide.
2. The complex of claim 1 , wherein the peptide acceptor is puromycin.
3. The complex of claim 1 , wherein the first high affinity ligand is biotin.
4. The complex of claim 1 , wherein the second high affinity ligand is biotin.
5. The complex of claim 1 , wherein the first and second high affinity ligands are biotin.
6. The complex of claim 1 , wherein the multimeric ligand acceptor protein is streptavidin.
7. The complex of claim 1 , wherein the polypeptide further comprises an affinity tag.
8. The complex of claim 7 , wherein the affinity tag is a FLAG, myc, histidine, or HA tag.
9. The complex of claim 1 , wherein the nucleic acid molecule comprises a source tag.
10. The complex of claim 1 , wherein the polypeptide is selected from the group consisting of an antibody, a VH domain, a VL domain, a Fab fragment, a single chain antibody, a nanobody, a unibody, an adnectin, an affibody, a DARPin, an anticalin, an avimer, a 10 Fn3 domain, and a versabody.
11. The complex of claim 1 , wherein the nucleic acid molecule is selected from the group consisting of ssRNA, ssDNA, ssDNA/RNA hybrid dsDNA, and dsDNA/RNA hybrid.
12. The complex of claim 1 , wherein the nucleic acid molecule does not contain an in-frame stop codon 3′ of the polypeptide-encoding sequence.
13. The complex of claim 1 , wherein the complex does not contain a ribosome.
14. The complex of claim 1 , wherein the peptide acceptor is bound to the second high affinity ligand through a linker.
15. The complex of claim 14 , wherein the linker comprises polyethylene glycol.
16. The complex of claim 14 , wherein the linker comprises polysialic acid.
17. An X-display complex comprising:
(a) a nucleic acid molecule comprising a polypeptide-encoding sequence, wherein the 3′ region of the nucleic acid molecule is covalently bound to a first biotin molecule;
(b) a polypeptide encoded by the nucleic acid molecule, wherein the polypeptide is covalently linked at the C-terminal residue through a peptide acceptor to a second biotin molecule, and wherein the peptide acceptor is selected from the group consisting of puromycin, pyrazolopyrimidine, phenylalanyl-adenosine, tyrosyl adenosine, alanyl adenosine, phenylalanyl 3′ deoxy 3′ amino adenosine, alanyl 3′ deoxy 3′ amino adenosine, and tyrosyl 3′ deoxy 3′ amino adenosine; and
(c) a single multimeric biotin-binding protein, wherein the biotin-binding protein is bound noncovalently and directly to both the first and second biotin molecules at distinct ligand binding sites on the biotin-binding protein, thereby noncovalently linking the nucleic acid molecule and the polypeptide.
18. The complex of claim 17 , wherein the polypeptide is selected from the group consisting of an antibody, a VH domain, a VL domain, a Fab fragment, a single chain antibody, a nanobody, a unibody, an adnectin, an affibody, a DARPin, an anticalin, an avimer, a 10 Fn3 domain, and a versabody.
19. The complex of claim 17 , wherein the nucleic acid molecule is selected from the group consisting of ssRNA, ssDNA, ssDNA/RNA hybrid dsDNA, and dsDNA/RNA hybrid.
20. The complex of claim 17 , wherein the nucleic acid molecule does not contain an in-frame stop codon 3′ of the polypeptide-encoding sequence.
21. The complex of claim 17 , wherein the complex does not contain a ribosome.
22. The complex of claim 17 , wherein the peptide acceptor is bound to the second high affinity ligand through a linker.
23. The complex of claim 22 , wherein the linker comprises polyethylene glycol.
24. The complex of claim 22 , wherein the linker comprises polysialic acid.
25. The complex of claim 17 , wherein the peptide acceptor is puromycin.
26. The complex of claim 17 , wherein the multimeric biotin-binding protein is streptavidin.
27. The complex of claim 17 , wherein the polypeptide further comprises an affinity tag.
28. The complex of claim 27 , wherein the affinity tag is a FLAG, myc, histidine, or HA tag.
29. The complex of claim 17 , wherein the nucleic acid molecule comprises a source tag.