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Affinity Selection of Phage
Overview
Each affinity-selection step starts with a mixture of phage and seeks to select phage whose displayed peptide binds a specific target receptor (see Protocol ID#2163). Phage are "captured" by binding to a receptor immobilized on a solid surface (e.g., a plastic petri dish). Unbound phage are washed away and bound phage are subsequently eluted (still in infective form), yielding a selected subset of the original phage mixture-the "eluate." The eluate from the first round of selection is amplified by infecting the phage into fresh cells, and the amplified eluate is then used as "input phage" for another round of selection. Altogether, two or three rounds of selection usually suffice to select for good binders-assuming, of course, the initial library contains such binders.
Procedure
A. Considerations of Stringency versus Yield
1. Yield is a very important consideration in the first round of selection (see below); in later rounds, yield may be sacrificed in the interest of interaction stringency (see Hint #8 and #9).
2. The input phage in the first round of selection comprises all clones in the initial library. Since the library is made up of many independent clones, each clone is represented by a small number of particles (approximately 100 transducing units (TU)/clone in a typical experiment). Consequently, if the yield of a particular clone is not high in the first round (greater than 1% for example), that clone may be lost. In practice, whenever possible, the authors of this protocol carry out the first round of selection using 10 μg of biotinylated receptor in the "one-step" selection procedure described below; this procedure gives the highest yields.
3. The entire eluate from the first round of selection should be amplified to create the input for the second round. Once the phage are amplified, an individual clone is represented by millions of phage particles. Thus, only a small portion of the amplified first eluate need be used as input for the second round of selection. Similarly, each clone in the unamplified eluates from the second and subsequent rounds of selection is represented by tens of thousands of phage particles (if not more), and no clones will be lost if only a portion of such an eluate is amplified.
B. Capture of phage using a Biotinylated Receptor
1. If the receptor protein is available in relatively pure form, it is convenient to biotinylate it at an accessible amino group (see Protocol ID#2188). This allows the receptor to be rapidly and irreversibly captured on streptavidin-coated petri dishes or ELISA dish wells under non-denaturing conditions, and also facilitates the ELISA assay (see Protocol ID#2167 and Protocol ID#2162).
2. The biotinylated receptor can be used in two ways, as detailed in Sections D and E. In the "one-step" selection (Section D) the phage are captured by biotinylated receptor that has been previously immobilized on the surface of a streptavidin-coated petri dish. In the "two-step" selection (Section E), phage are first allowed to bind with the biotinylated receptor in solution, subsequently captured on the streptavidin-coated dish.
C. Input Phage
1. For either one- or two-step selection, use approximately 1 to 3 X1011 TU of input phage. This can be a 10 μl portion of an initial library at a physical particle concentration of 2 X 1014 virions/ml, or a 100 μl portion of an amplified eluate from a prior round of affinity selection at a physical particle concentration of approximately 5 X 1013 virions/ml (see Protocol ID#2175). The input phage need not be purified (see Hint #10).
D. One-Step Selection
1. Coat a 35 mm petri dish with 400 μl of 10 μg/ml Streptavidin Solution for at least 1 hr at room temperature.
2. Aspirate the Streptavidin Solution and fill the dish to the brim with Blocking Solution. Leave the plates with the lids removed for 2 hr at room temperature.
3. Remove the Blocking Solution (see Hint #11). Wash the plate five times with TBS/Tween from a squirt-bottle, emptying the dish by aspiration each time.
4. Add the desired amount of biotinylated receptor (0.01 to 10 μg; see Hint #12), in 400 μl of TTDBA; allow the receptor to bind to the dish for at least 2 hr at 4°C (see Hint #13).
5. Wash the dish five times with TBS/Tween as in Step #3 so as to remove unbound receptor, and fill the dish with 400 μl of TTDBA.
6. Add 4 μl of Biotin solution and rock gently at room temperature for 10 min so as to block unoccupied biotin-binding sites on the immobilized streptavidin.
7. Add the input phage (see "Input phage" in the introduction) and rock the dish gently for 4 hr at 4°C. There is no need to remove excess free biotin.
8. Wash the dish ten times with TBST/Tween as in Step #3.
9. Elute the bound phage from the dish with 400 μl of Elution Buffer for 10 min on a rocker
10. Transfer the eluate to a microcentrifuge tube and neutralize it by mixing it with 75 μl of 1 M Tris-HCl (pH 9.1). The Blocking Solution (which is yellow due to the presence of Phenol Red) will turn reddish purple upon neutralization. If this is not the last round of selection, amplify this eluate as detailed in Section F. If it is the last eluate, titer and propagate individual clones as detailed in Section G.
E. Two-Step Selection (see Hint #14)
1. Equilibrate the input phage (see Section C) overnight at 4°C with the desired amount of biotinylated receptor (usually less than 1 μg) in TTDBA (approximately 100 μl).
2. Coat a 35 mm petri dish with 400 μl of 10 μg/ml Streptavidin Solution for at least 1 hr at room temperature.
3. Aspirate the Streptavidin Solution and fill the dish to the brim with Blocking Solution. Leave the plates with the lids removed for 2 hr at room temperature.
4. Remove the Blocking Solution (see Hint #11). Wash the plate five times with TBS/Tween from a squirt-bottle, emptying the dish by aspiration each time.
5. Fill the dish with 400 μl of TTDBA.
6. Add the reaction mixture (Step #1, Section E) to the Streptavidin-coated dish from the previous step. Rock the dish gently for 10 min at room temperature to permit capture by immobilized Streptavidin.
7. Wash the dish ten times with TBST/Tween as in Step #3.
8. Elute the bound phage from the dish with 400 μl of Elution Buffer for 10 min on a rocker.
9. Transfer the eluate to a microcentrifuge tube and neutralize it by mixing it with 75 μl of 1 M Tris-HCl (pH 9.1). The Blocking Solution (which is yellow due to the presence of Phenol Red) will turn reddish-purple upon neutralization. If this is not the last round of selection, amplify this eluate as detailed in Section F. If it is the last eluate, titer and propagate individual clones as detailed in Section G.
F. Quantifying Yield and Amplification of Eluates (see Hint #15)
1. (For first-round eluates only) Concentrate the entire first-round eluate (using a Centricon 30-KDa ultrafilter [Amicon]) and wash once with TBS by centrifuging at 5,000 rpm in the Sorvall SS34 rotor (3,000 X g) using thick-walled rubber adaptors to produce a final volume of approximately 100 μl.
2. Mix 100 μl of eluate (the entire eluate if from the first round) and 100 μl of starved cells (see Protocol ID#2173, Section A) or terrific broth culture (see Protocol ID#2173, Section B) in a 1.5 ml microcentrifuge tube.
3. Incubate the tube for 10 to 30 min at room temperature.
4. Pipette the infected cells into a 125 ml culture flask containing 20 ml of NZY with 0.2 μg/ml Tetracycline Glycerol Stock.
5. Shake the culture for 30 to 60 min at 37°C.
6. Spread 200 μl portions of appropriate serial dilutions (see Protocol ID#2181) of the culture (diluent = NZY) on NZY plates containing 40 μg/ml Tetracycline to quantify the output of the affinity selection (see Hint #16).
7. At the same time, titer a suitable serial dilution (see Protocol ID#2181) of the input phage (diluent = TBS/Gelatin; dilution should aim at approximately 105 TU/ml, equivalent to approximately 2 X 106 physical particles/ml) by the ordinary analytical titering method ( see Protocol ID#2173 Section C ) on the same starved cells or terrific broth culture as were used in Section F Step #2); the yield of each affinity selection can be calculated by dividing the output by the input (see Hint #17).
8. Meanwhile, add 20 μl of 20 mg/ml Tetracycline to the 20 ml culture to bring the total concentration of Tetracycline to 20 μg/ml.
9. Continue shaking the culture overnight at 37°C.
10. Centrifuge the culture at 8,000 rpm in a SS34 rotor (7,500 X g) for 10 minutes at 4°C.
11. Remove the supernatant to a fresh centrifuge tube (Oak Ridge) and centrifuge the culture at 5,000 rpm in a SS34 rotor (3,000 X g) for 10 minutes at 4°C.
12. Pour the doubly cleared supernatant (approximately 20 ml) into a fresh centrifuge tube.
13. Add 3 ml of PEG/NaCl and mix by many inversions; allow the phage to precipitate overnight in the refrigerator.
14. Centrifuge the sample at 10,000 rpm in a SS34 rotor (12,000 X g) for 10 min at 4°C to collect the precipitated phage.
15. Decant the supernatant by aspiration.
16. Orient the tube as in the previous centrifugation step, and centrifuge the sample at 10,000 rpm in a SS34 rotor (12,000 X g) for 5 min at 4°C to remove the residual supernatant.
17. Remove any residual supernatant by aspiration.
18. Dissolve the pellet in 1 ml of TBS and transfer the solution to a 1.5 ml microcentrifuge tube.
19. Centrifuge the sample in a microcentrifuge for 1 min at top speed to clear undissolved material, transferring the supernatant to a second 1.5 ml microcentrifuge tube.
20. Add 150 μl of PEG/NaCl, vortex to mix, and place at 4°C for a minimum of 1 hr.
21. Centrifuge in a microcentrifuge for 5 min at maximum speed.
22. Decant the supernatant by aspiration.
23. Orient the tube as in the previous centrifugation step, and centrifuge in a microcentrifuge for 2 to 3 min at maximum speed to remove the residual supernatant.
24. Remove any residual supernatant by aspiration.
25. Dissolve the pellet in 200 μl of TBS containing 0.2% NaN3.
26. Centrifuge in a microcentrifuge for 1 min at top speed to clear undissolved material.
27. Transfer the supernatant to a 0.5 ml microcentrifuge tube. This is the amplified eluate; the physical particle concentration should be approximately 5 X 1013 virions/ml, regardless of the titer in the unamplified eluate; the titer is approximately 0.5 to 5 X 1012 TU/ml. Usually 40 to 100 μl of this amplified eluate is used for the next round of affinity selection.
G. Quantifying Yield and Propagating Clones from the Final Eluate (see Hint #18)
1. Using TBS/Gelatin as the diluent, make suitable serial dilutions (see Protocol ID#2181) of both the input (see Hint #17) and the unamplified final eluate (see Hint #19).
24. Titer these dilutions on starved cells (see Protocol ID#2173).
25. These clones may now be propagated and processed (see Protocol ID#2169) for individual characterization.
Solutions
NaN3 (5% stock)
TBS/Tween
Prepare in 1X TBS
0.5% (v/v) Tween-20
TBS/Gelatin
After autoclaving, swirl to mix in the melted Gelatin
Store at room temperature
Autoclave 0.1 g Gelatin in 100 ml of TBS
NaN3 (5% stock)
TBS (1X)
50 mM Tris HCl, pH 7.5
150 mM NaCl
Autoclave and store at room temperature (see Hint #1)
NaN3 (5% stock)
Place in 15 ml bottle, store in refrigerator, with appropriate warning on tube
Dissolve 0.5 g solid NaN3 in 9.5 ml of ddH2O (do not autoclave) (CAUTION! See Hint #7)
Blocking Solution
0.1 M NaHCO3
0.1 μg/ml Streptavidin
5 mg/ml dialyzed Bovine Serum Albumin (BSA)
0.02% NaN3
PEG/NaCl (16.7%/3.3 M stock)
Stir until solutes dissolve (see Hint #5)
116.9 g NaCl
Prepare in 475 ml of ddH2O (the total volume should be 600 ml)
100 g PEG 8000 (see Hint #4)
Store at 4°C (see Hint #6)
10 μg/ml Streptavidin
Prepare in 0.1 M NaHCO3
Tetracycline Glycerol Stock
Allow to cool to room temperature
Mix thoroughly
Filter sterilize 40 ml of Tetracycline Stock into the Glycerol
Autoclave 40 ml of Glycerol
Store at 20°C away from the light
Tetracycline Stock
20 mg/ml Tetracycline, prepared in ddH2O
Filter sterilize
NZY medium (1X)
Adjust pH to 7.5 with NaOH
5 g NaCl
Autoclave and store at room temperature
Dissolve in 1 liter of ddH2O
Also see Hint #3
5 g Yeast Extract
10 g NZ Amine A
Elution buffer
0.1 M HCl, pH adjusted to 2.2 with Glycine (see Hint #2)
1 mg/ml BSA
0.1 mg/ml Phenol Red
Biotin (10 mM stock)
10 mM Biotin
Add 1 M NaOH dropwise with constant stirring
Prepare in ddH2O
TTDBA
Prepare in 1X TBS
0.5% (v/v) Tween-20
1 mg/ml BSA
0.02% NaN3
BioReagents and Chemicals
NZ Amine A
Tetracycline
Streptavidin
Elution Buffer
Sodium Hydroxide
Phenol Red
Bovine Serum Albumin
Glycerol
Hydrochloric Acid
Sodium Nitrate
Tween-20
Tris-Cl
Sodium Bicarbonate
Yeast Extract
PEG 8000
Biotin
Glycine
Gelatin
Sodium Chloride
Protocol Hints
1. The contributors of this protocol suggest making a 10X stock that can be stored at room temperature without autoclaving.
2. The Elution Buffer is prepared and adjusted as a 4X stock, filter-sterilized, and stored at room temperature. The phenol red gives a visual indication of whether the pH is (roughly) correct after the eluate is neutralized.
3. The contributors use this rich media routinely in the laboratory. Other media, such as LB, can also be used. NZY has the advantage that NZ amine A (from Humko Sheffield Chemical, P.O. Box 630, Norwich, NY 13815) is much cheaper than tryptone.
4. Purchased from Union Carbide and was previously called PEG 6000.
5. It may be necessary to heat the solution to 65°C briefly to dissolve the last crystals of PEG.
6. This solution can be autoclaved; as it cools, shake it occasionally to prevent separation into two phases. If separation does occur, autoclave again to homogenize the solution.
7. CAUTION! This substance is a biohazard. Consult this agent's MSDS for proper handling instructions.
8. There is always a small amount of non-specifically bound phage. If stringency is set too high, the yield of specifically bound phage will fall below the background of non-specifically bound phage, and the discrimination in favor of high affinity will be lost.
9. In practice, because the relationship between selection conditions and stringency is unknown in advance, it is recommended to explore a range of conditions in the later rounds of selection. Use varying amounts of biotinylated receptor (less than 1 μg) in both the one- and two-step selection procedures described below.
10. The contributors have successfully used the culture supernatant from eluate amplification directly as the input to the next round of affinity selection.
11. Pour the blocking solution back into its container (it may be re-used until it appears cloudy)
12. The latter amount is recommended in the first round.
13. When the amount of biotinylated receptor added at step 4 below is enough to saturate the immobilized streptavidin (1 to 10 μg per 35 mm dish), this procedure gives the maximum achievable yield, which can reach 20% of the input phage. When each phage particle displays multiple copies of the random peptide, as in most of the contributor's libraries, this high yield is plausibly attributed to attachment of a single virion to two or more neighboring receptor molecules (or, in the case of antibodies, to both Fab domains of a single IgG molecule); a particle captured multivalently in this fashion may dissociate from the solid surface exceedingly slowly, even if the underlying monovalent affinity is only modest. As the density of an immobilized monovalent receptor is decreased, this "avidity effect" is reduced, possibly to the point where yield from monovalent attachment comes to dominate the output-conditions that should strongly favor high affinity.
14. During the equilibrium step (Section E, Step #1), monovalent receptors bind phage reversibly according to solution-phase equilibrium kinetics. If there is little dissociation and reassociation during the subsequent 10 min capture step (Section E, Step #6), the situation at the beginning of the capture step will largely determine the relative yields of different clones. If, at the other extreme, receptors dissociate and reassociate very rapidly during the capture step, two-step selection is really equivalent to an abbreviated one-step selection. If desired, reassociation can be suppressed during the capture step by adding a competitive ligand for the receptor at a high concentration. In practice, two-step selection gives considerably lower yields than one-step selection, even when reassociation is not suppressed.
15. Eluates that are to serve as input for further rounds of affinity selection are amplified by propagating the phage in fresh host cells. The eluate from the first round is first concentrated (Section F, Step #1) to permit the entire eluate to be amplified (see Section A for considerations). There is no need to concentrate eluates from subsequent rounds as each clone is represented by multiple phage particles.
16. Assuming the input to the affinity selection was approximately 1011 TU, as recommended, dilutions in the range of 10-1to 10-4 will cover yields in the range of 3 X 10-5% (typical non-specific background) to 1% (the highest yields we ordinarily observe). For the first round, the 10-1and 10-2dilutions will almost always suffice.
17. A suitable dilution of an initial library with a physical particle concentration of, say, 2 X 10-14 virions/ml (~1013 TU/ml) is 10-8; suitable dilutions of an amplified eluate with a physical particle concentration of ~5 X 1013 virions/ml are 10-7 and 10-8.
18. There is usually no need to amplify the final eluate. Instead, yield is determined by analytical titering of suitable dilutions of both input and output (eluate). The colonies from the output titering also serve as individual output clones that are propagated and characterized individually by sequencing and binding assays such as ELISA.
19. Assuming an input of 1011 TU, suitable dilutions of the output (unamplified eluate; see Section D Step #10; volume 475 μl) are 10-1 to 10-5. This will cover yields up to approximately 5%.
