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MOLECULAR BIOLOGY: WORKING WITH DNA

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"Low Background" Phage Display Protocol for the Isolation of Protein-Binding Peptides

"Low Background" Phage Display Protocol for the Isolation of Protein-Binding Peptides Title | Overview | Procedure | Solutions | BioChemicals | Hints | Printable Version
Contributor:	The Laboratory of Tom Kodadek at the University of Texas, Southwestern Medical Center
Overview Title | Overview | Procedure | Solutions | BioChemicals | Hints | Printable Version
Phage display is a convenient method to select polypeptides that bind to a molecule of interest. The following method is designed to reduce or eliminate the isolation of "background" phage (peptide-displaying phage that bind the solid support or some other spurious target). This is accomplished through several rounds of panning using different forms of the target protein and different solid supports. Thus, the only constant element presented to the phage is the target protein itself. Using a library of approximately 1 X 109 20-mer oligonucleotides displayed at the N-terminus of the pIII protein of M13 (kindly provided by Dr. W. Dower at Affymax), the contributors of this protocol routinely isolate 2 to 10 peptides, all of which bind the target protein with a KD of between 1 X 10-7 M and 5 X 10-6 M. In cases where specificity of the phage binding has been analyzed,the specificity of interaction has been found to be very high (for an example, see Citation #1).
Procedure Title | Overview | Procedure | Solutions | BioChemicals | Hints | Printable Version
A. Phage Selection Round One All steps are carried out at room temperature. 1. Add 200 μl of His6-Tagged Bait Protein (approximately 10 μg of protein) to each well of an ELISA plate. 2. Seal the plate with parafilm to avoid evaporation and incubate at room temperature for 2 hours. 3. Aspirate the solution from each ELISA plate well. 4. Block each well by adding 200 μl of 1% BSA Solution. 5. Re-seal the plate to avoid evaporation and incubate at room temperature for 2 hr. 6. Aspirate the solution from each ELISA plate well. 7. Add 500 μl of PBS/Tween Solution and aspirate the solution from each well. 8. Repeat Step #A6 three more times (for a total of four washes). 9. Add approximately 1 X 1010 phage prepared in PBS/Tween Solution (see Hint #1). 10. Seal the plate with parafilm to avoid evaporation and incubate at room temperature for 2 hours. 11. Aspirate the solution from each ELISA plate well. 12. Wash each ELISA well eight times with PBS/Tween Solution (Steps #A7 to #A8). 13. Add 100 μl of 50 mM Glycine, pH 2.0 to each ELISA well and incubate for 15 min. 14. Transfer the Glycine solution to a 500 μl microcentrifuge tube. 15. Add 25 μl of 1 M Tris to neutralize the solution. 16. Apply the collected supernatant to an appropriate amount of cells to amplify the selected phage (such as K91 cells, also see Hint #1). 17. Collect the resulting phage particles and continue with Section B. B. Phage Selection Round Two 1. Add 20 μl of Ni-NTA Beads to a 1.5 ml microcentrifuge tube. 2. Add 100 μl of His6-Tagged Bait Protein (approximately 10 μg of protein) to the microcentrifuge tube (see Hint #2). 3. Incubate for 1 hr at 4°C on a rotating mixing wheel. 4. Centrifuge at 2,000 rpm in a microcentrifuge for 1 min to pellet the Ni-NTA Beads and aspirate the supernatant. 5. Add 1 ml of 50 mM Na2HPO4, pH 8.0 buffer at 4°C and mix gently. 6. Aspirate the supernatant. 7. Wash the Ni-NTA beads three more times (Steps #B4 to #B6). 8. Add 1 X 1010 phage prepared in 50 mM Na2HPO4, pH 8.0. 9. Incubate for 1 hr at 4°C on a rotating mixing wheel. 10. Centrifuge at 2,000 rpm in a microcentrifuge for 1 min to pellet the Ni-NTA Beads and aspirate the supernatant. 11. Add 1 ml of 50 mM Na2HPO4, pH 8.0 buffer at 4°C and mix gently. 12. Aspirate the supernatant. 13. Wash the Ni-NTA beads four more times (Steps #B10 to #B12). 14. Add 100 μl of 200 mM Imidozole and incubate for 10 min at room temperature. 15. Centrifuge at 2,000 rpm in a microcentrifuge for 1 min to pellet the Ni-NTA Beads and aspirate the supernatant. 16. Apply the collected supernatant to an appropriate amount of cells to amplify the selected phage (such as K91 cells; see Hint #1). 17. Collect the resulting phage particles and continue with Section C. C. Phage Selection Rounds Three and Four 1. Add 20 μl of Glutathione-Sepharose beads (Amersham/Pharmacia) to a 1.5 ml microcentrifuge tube. 2. Add 10 μg of GST-Fused Bait Protein prepared in 100 μl of PBS/Triton. 3. Incubate for 30 min at 4°C on a rotating mixing wheel. 4. Centrifuge the GST-Sepharose Reaction at 2,000 rpm in a microcentrifuge for 1 min to pellet the Glutathione-Sepharose beads and aspirate the supernatant. 6. Add 1 ml of PBS/Triton to the GST-Sepharose Reaction, mix gently, centrifuge at 2,000 rpm in a microcentrifuge for 1 min to pellet the Glutathione-Sepharose beads, and aspirate the supernatant. 7. Wash the Glutathione-Sepharose beads two more times (Step #D6). 8. Combine equal volumes of isolated Phage particles with collected Glutathione-Sepharose beads. 9. Incubate for 2 hr at 4°C on a rotating mixing wheel. 10. Centrifuge the reaction at 2,000 rpm in a microcentrifuge for 1 min to pellet the Glutathione-Sepharose beads and aspirate the supernatant. 11. Add 1 ml of PBS/Triton and mix gently. 12. Centrifuge the reaction at 2,000 rpm in a microcentrifuge for 1 min to pellet the Glutathione-Sepharose beads and aspirate the supernatant. 13. Repeat the PBS/Triton wash three more times (Steps #C11 to #C13). 14. Add 300 μl of TEV Protease Buffer and mix gently. 15. Centrifuge the reaction at 2,000 rpm in a microcentrifuge for 1 min to pellet the Glutathione-Sepharose beads and aspirate the supernatant. 16. Repeat the TEV Protease Buffer wash one more time (Steps #C14 to #C15). 17. Add 100 μl of TEV buffer to the washed Sepharose beads and 3 μl of TEV Protease (Tobacco Etch Virus Protease, 30 Units). 18. Elute the bound phage by incubating for 30 min at 30°C on a rotating mixing wheel. 19. Centrifuge the reaction at 2,000 rpm in a microcentrifuge for 1 min to pellet the Sepharose beads. 20. Apply the collected supernatant to an appropriate amount of cells to amplify the selected phage (such as K91 cells). 21. Isolate the resulting phage and repeat Section C one more time. D. Phage Selection Rounds Five and Six Preparation of Sepharose Treated Phage (prepare in parallel with Binding Subsection below) 1. Add 50 μl of Glutathione-Sepharose beads to a 1.5 ml microcentrifuge tube. 2. Add 1 X 1010 phage prepared in 200 μl of PBS/Triton. 3. Incubate for 30 min at 4°C on a rotating mixing wheel. 4. Centrifuge at 2,000 rpm in a microcentrifuge for 1 min to pellet the Glutathione-Sepharose beads and collect the supernatant. 5. Combine the collected Phage supernatant with Glutathione-Sepharose Beads as described in Step #D13. Binding of Glutathione-Sepharose Beads to GST-Fused Bait Protein (prepare in parallel with Phage Treatment above) 6. Add 20 μl of Glutathione-Sepharose beads to a 1.5 ml microcentrifuge tube. 7. Add 10 μg of GST-Fused Bait Protein prepared in 100 μl of PBS/Triton. 8. Incubate for 30 min at 4°C on a rotating mixing wheel. 9. Centrifuge the GST-Sepharose Reaction at 2,000 rpm in a microcentrifuge for 1 min to pellet the Glutathione-Sepharose beads and aspirate the supernatant. 10. Add 1 ml of PBS/Triton to the GST-Sepharose Reaction, mix gently, centrifuge at 2,000 rpm in a microcentrifuge for 1 min to pellet the Glutathione-Sepharose beads, and aspirate the supernatant. 11. Wash the Sepharose beads from the GST-Sepharose Reaction two more times (Step #D10). 12. Combine the collected Glutathione-Sepharose beads with Phage supernatant as described in Step #D13. Phage and Sepharose Bead Incubation 13. Combine the prepared Phage supernatant (prepared in Steps #D1 to #D5) with collected Glutathione-Sepharose beads (prepared in Steps #D6 to #D12). 14. Incubate for 2 hr at 4°C on a rotating mixing wheel. 15. Centrifuge the reaction at 2,000 rpm in a microcentrifuge for 1 min to pellet the Glutathione-Sepharose beads and aspirate the supernatant. 16. Add 1 ml of High Stringent Wash Solution and mix gently. 17. Centrifuge the reaction at 2,000 rpm in a microcentrifuge for 1 min to pellet the Glutathione-Sepharose beads and aspirate the supernatant. 18. Repeat the high-stringent wash three more times (Steps #D16 to #D17). 19. Add 300 μl of TEV Protease Buffer and mix gently. 20. Centrifuge the reaction at 2,000 rpm in a microcentrifuge for 1 min to pellet the Glutathione-Sepharose beads and aspirate the supernatant. 21. Repeat the TEV Protease Buffer wash one more time (Steps #19 to #D20). 22. Add 100 μl of TEV buffer to the washed Glutathione-Sepharose beads and 3 μl of TEV Protease (Tobacco Etch Virus Protease, 30 Units). 23. Elute the bound phage by incubating for 30 min at 30°C on a rotating mixing wheel. 24. Centrifuge the reaction at 2,000 rpm in a microcentrifuge for 1 min to pellet the Glutathione-Sepharose beads. 25. Apply the collected supernatant to an appropriate amount of cells to amplify the selected phage (such as K91 cells). 26. Isolate the resulting phage and repeat Section D one more time.
Solutions Title | Overview | Procedure | Solutions | BioChemicals | Hints | Printable Version
200 mM Imidozole TEV Protease Buffer    Supplied with TEV Protease (Life Technologies, Inc) High Stringency Wash    1% (v/v) Triton X-100 550 mM NaCl Prepared in PBS PBS/Triton    1% (v/v) Triton X-100 Prepare in PBS 50 mM Na2HPO4, pH 8.0 50 mM Na2HPO4, pH 8.0 50 mM Na2HPO4, pH 8.0    50 mM Sodium Phosphate, Dibasic (Na2HPO4), pH 8.0 50 mM Sodium Phosphate, Dibasic, pH 8.0 100 mM NaHCO3, pH 8.5    100 mM Sodium Bicarbonate (NaHCO3), pH 8.5 50 mM Na2HPO4, pH 8.0 Ni-NTA Beads    Ni-NTA-Agarose beads from Qiagen 1 M Tris 50 mM Glycine, pH 2.0    50 mM Glycine-HCl, pH 2.0 PBS/Tween Solution    0.1% (v/v) Tween 20 Prepared in PBS PBS    4.3 mM Sodium Phosphate, Dibasic (Na2HPO4) 137 mM Sodium Chloride (NaCl) 2.7 mM Potassium Chloride (KCl) See Protocol ID#2152 for detailed preparation 1.4 mM Potassium Phosphate, Monobasic (KH2PO4) 1% BSA Solution    Prepared in 100 mM NaHCO3, pH 8.5 1% (w/v) Bovine Serum Albumin (BSA) His6-Tagged Bait Protein His6-Tagged Bait Protein His6-Tagged Bait Protein    Prepared in 100 mM NaHCO3, pH 8.5 Approximately 20 μg/μl final concentration
BioReagents and Chemicals Title | Overview | Procedure | Solutions | BioChemicals | Hints | Printable Version
Glycine-HCl Tween 20 Tris TEV Protease Buffer Bovine Serum Albumin Sodium Bicarbonate TEV Protease Ni-NTA-Agarose Beads Imidozole Sodium Phosphate, Dibasic Potassium Chloride Potassium Phosphate, Monobasic Sodium Chloride Triton X-100
Protocol Hints Title | Overview | Procedure | Solutions | BioChemicals | Hints | Printable Version
1. Use a method for phage purification that is specific to the phage currently in use or adapt Protocol ID#131. 2. GST fusion proteins were constructed by cloning the gene encoding the protein of interest into pGEXcs (Citation #2). This results in a fusion protein in which a TEV protease site is located between the GST and the protein of interest, allowing for TEV protease-mediated release of the bound phage.
Citation and/or Web Resources Title | Overview | Procedure | Solutions | BioChemicals | Hints | Printable Version
2. Parks, T.D., Leuther, K.K., Howard, E.D., Johnston, S.A., and Dougherty, W.G. Release of proteins and peptides from fusion proteins using a recombinant plant virus proteinase. Anal. Biochem 1994;216:413-417. 1. Han, Y. and Kodadek, T. Peptides selected to bind the Gal80 repressor are potent transcriptional activation domains in yeast. J. Biol. Chem. 2000;275:14979-14984.