Methods of making and utilizing amber-obligated phage display libraries
Embodiments of the present disclosure pertain to methods of constructing a phage display library where at least 90% of combinatorial regions in the phage display library include at least one inframe amber codon. Further embodiments of the present disclosure pertain to the formed phage display libraries. Additional embodiments of the present disclosure pertain to methods of selecting peptides or proteins that bind to a desired target (e.g., a ligand binding site of a desired target) by utilizing the phage display libraries of the present disclosure. Further embodiments of the present disclosure pertain to peptides that have been screened from the phage display libraries and methods of the present disclosure, such as inhibitors of sirtuin 2, or inhibitors of ENL.
1. A method of constructing a phage display library, said method comprising:
(a) providing a naïve phage display library,
wherein the naïve phage display library is a heptapeptide library that comprises a plurality of nucleic acids that encode a phage coat protein gene,
wherein the phage coat protein gene comprises seven randomized (NNK) codons fused to its N-terminus, and
wherein the combinatorial regions of at least some of the nucleic acids comprise at least one in-frame amber codon;
(b) transforming the naïve phage display library into bacterial host cells;
(c) inducing expression of the phage coat protein gene in the transformed bacterial host cells,
wherein the expression of the phage coat protein from nucleic acids that only contain in-frame sense codons in the phage coat protein gene renders the bacterial host cells immune to infection by a helper phage;
(d) infecting the transformed bacterial host cells with the helper phage,
wherein the helper phage encodes and expresses a gene that renders the infected bacteria resistant to an antibiotic;
(e) growing the infected and uninfected bacterial host cells in a medium containing the antibiotic,
wherein the growing results in the selection of the infected bacteria, and
wherein the infected bacteria comprise the helper phage and at least some of the nucleic acids comprising the phage coat protein gene with the at least one in-frame amber codon in the combinatorial region;
(f) extracting the nucleic acids from the bacterial cell host cells;
(g) transforming the extracted nucleic acids into amber-suppressing bacterial host strains,
wherein the amber-suppressing bacterial host strains are co-infected with a knockout helper phage that does not express the phage coat protein gene, and
wherein the transforming allows for the selective production of phage particles from cells harboring nucleic acids comprising the phage coat protein gene with the at least one in-frame amber codon in the combinatorial region; and
(h) purifying the nucleic acids from the produced phage particles to provide the phage display library,
wherein at least 90% of the combinatorial regions in the phage display library comprise at least one in-frame amber codon.
2. The method of claim 1 , wherein more than 90% of the combinatorial regions in the phage display library comprise at least one in-frame amber codon.
3. The method of claim 1 ,
wherein the naïve phage display library is prepared by site-directed mutagenesis,
wherein the phage display library comprises a plurality of phages, phagemids, or combinations thereof, and wherein the plurality of nucleic acids in the phage display library comprise the nucleic acids of the phages or phagemids,
wherein the combinatorial region encodes a peptide or a protein, and
wherein the combinatorial region in at least some of the nucleic acids in the phage display library comprise a plurality of in-frame amber codons, wherein the plurality of in-frame amber codons are randomly distributed throughout the combinatorial region.
4. The method of claim 1 ,
wherein the bacterial host cells comprise E. coli bearing an F sex pilus,
wherein the phage coat protein gene is the PIII gene,
wherein the phage coat protein gene is positioned near an IPTG-inducible promoter, and wherein the phage coat protein is expressed by exposing the bacterial host cells to IPTG,
wherein the helper phage is a CM13 helper phage, and
wherein the helper phage encodes and expresses a gene that renders the infected bacteria resistant to kanamycin.