Methods of constructing libraries of genetic packages that collectively display the members of a diverse family of peptides, polypeptides or proteins
View Patent ↗Methods useful in constructing libraries that collectively display members of diverse families of peptides, polypeptides or proteins and the libraries produced using those methods. Methods of screening those libraries and the peptides, polypeptides or proteins identified by such screens.
1. A method for preparing a nucleic acid, the method comprising the steps of:
(i) amplifying a nucleic acid that encodes a polypeptide using a primer complementary to at least part of a synthetic sequence located at the 5′ terminus of the nucleic acid sequence;
(ii) rendering the amplified nucleic acid obtained from step (i) single-stranded;
(iii) hybridizing the single-stranded nucleic acid obtained from step (ii) with a single-stranded oligonucleotide to form a locally double-stranded region, wherein there is a single site for a restriction endonuclease within the locally double-stranded region;
(iv) forming a cleaved DNA complex by cleaving the locally double-stranded region formed by the hybridizing step (iii) with the restriction endonuclease at the single site for the restriction endonuclease to remove all unwanted 5′ nucleotides from the amplified nucleic acid; and
(v) cloning the cleaved DNA complex obtained from step (iv) a vector for expressing the polypeptide encoded by the nucleic acid;
wherein the hybridizing and the cleaving steps are performed at a temperature under which the single-stranded nucleic acid is maintained in substantially single-stranded form and associates with the single-stranded oligonucleotide to form the locally double-stranded region, and wherein the restriction endonuclease is active at the temperature.
2. The method according to claim 1 , wherein the nucleic acid encodes at least a portion of an immunoglobulin.
3. The method according to claim 2 , wherein the immunoglobulin comprises a Fab or single chain Fv.
4. The method according to claim 3 , wherein the immunoglobulin comprises at least a portion of a heavy chain.
5. The method according to claim 4 , wherein at least a portion of the heavy chain is human.
6. The method according to claim 3 , wherein the immunoglobulin comprises at least a portion of FR1.
7. The method according to claim 6 , wherein at least a portion of the FR1 is human.
8. The method according to claim 3 , wherein the immunoglobulin comprises at least a portion of a light chain.
9. The method according to claim 8 , wherein at least a portion of the light chain is human.
10. The method according to claim 2 , wherein the immunoglobulin comprises at least a portion of a heavy chain.
11. The method according to claim 10 , wherein at least a portion of the heavy chain is human.
12. The method according to claim 2 , wherein the immunoglobulin comprises at least a portion of FR1.
13. The method according to claim 12 , wherein at least a portion of the FR1 is human.
14. The method according to claim 2 , Wherein the immunoglobulin comprises at least a portion of a light chain.
15. The method according to claim 14 , Wherein at least a portion of the light chain is human.
16. The method according to claim 1 , wherein the nucleic acid is at least in part derived from a patient suffering from at least one autoimmune disease or cancer.
17. The method according to claim 16 , wherein the autoimmune disease is lupus erythematosus, systemic sclerosis, rheumatoid arthritis, antiphosolipid syndrome or vasculitis.
18. The method according to claim 16 , wherein the nucleic acid is at least in part isolated from peripheral blood cells, bone marrow cells, spleen cells or lymph node cells.
19. The method according to claim 1 , Wherein the temperature is between 45° C. and 75° C.
20. The method according to claim 19 , wherein the temperature is between 50° C. and 60° C.
21. The method according to claim 20 , wherein the temperature is between 55° C. and 60° C.
22. The method according to claim 1 , wherein the length of the single-stranded oligonucleotide is between 17 and 30 bases.
23. The method according to claim 22 , wherein the length of the single-stranded oligonucleotide is between 18 and 24 bases.
24. The method according to claim 1 , wherein the restriction endonuclease is selected from the group consisting of TaaI MacIII, Tsp45I, HphI, BsaJI, AluI, BlpI, DdeI, BglII, MslI, BsiEI, EaeI, EagI, HaeIII, Bst4Cl, HpyCH4III, HinfI, MlyI, PleI, MnlI, HpyCH4V, BsmAI, BpmI, XmnI, and Sad.
25. The method according to claim 24 , wherein the restriction endonuclease is selected from the group consisting of Bst4CI, TaaI, HpyCH4III, BlpI, HpyCH4V and MslI.
26. The method of claim 1 , wherein the vector is a. phage display vector.
27. The method of claim 1 , wherein the cloning step is performed using a partially duplexed synthetic DNA adapter.