Affinity Selection - University of Missouri



Affinity Selection

General considerations

Each affinity-selection step starts with a mixture of phage, and seeks to select from that mixture phage whose displayed peptide binds the target receptor, antibody or other binding molecule (we call such molecules generically “selectors”). These phage are specifically “captured” by immobilizing the selector on a solid surface (e.g., a plastic petri dish); unbound phage are washed away and captured phage are eluted (still in infective form), yielding a selected subset of the original phage mixture that is called an “eluate.” If a second round of affinity selection is planned, the eluate from the first round must be amplified by infecting the phage into fresh cells; the amplified eluate is then used as input to 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.

Stringency versus yield

The stringency of affinity selection is more or less opposed to yield, and is controllable in some degree by the choice of conditions, as will be detailed below. Yield is all-important in the first round of selection (see below), but in later rounds yield can be sacrificed in the interest of stringency.

There is a limit to stringency, however. The reason is that there is always a background yield of non-specifically bound phage; if stringency is set too high, the yield of specifically captured phage will fall far below the background of non-specifically bound phage, and all power of discrimination in favor of high affinity is lost.

In practice, because the relationship between selection conditions and stringency is unknown in advance, it is advisable to explore a range of conditions in the later rounds of selection. This usually means trying different amounts of biotinylated selector (1%, say), that clone has a good chance of being lost, and of course can never be recovered. In practice, where possible we carry out the first round using 10 µg biotinylated selector in the “one-step” selection procedure described below; this procedure gives the highest yields.

By the logic in the previous paragraph, it is also important that the entire eluate from the first round be amplified to create the input for the second round (see step 13 and the note above it). Once the phage are amplified, each clone is represented by millions of phage particles, and no clones will be lost by using only a portion of the amplified first eluate as input to 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.

Capture via a biotinylated selector: “one-step” and “two-step” selection

If selector protein is available in relatively pure form, it is convenient to biotinylate it at accessible amino groups, as described in biotinylation.doc. This allows it to be rapidly and irreversibly captured on streptavidin-coated petri dishes or ELISA dish wells under non-denaturing conditions, and also facilitates ELISA. (Numerous alternative immobilization methods are available, but won't be discussed here.)

The biotinylated selector can be used in two ways (details in the next two subsections): in “one-step” selection, phage are captured by biotinylated selector that has been pre-immobilized on the surface of a streptavidin-coated petri dish; while in “two-step” selection, phage are reacted with biotinylated selector in solution, then subsequently captured on a streptavidin-coated dish.

Input phage

The input to the first round of affinity selection is the initial phage display library; the input to the second and subsequent rounds of infection is the amplified eluate from the previous round.

The number of input phage must be large enough to adequately represent all the clones of interest—i.e., clones with high affinity for the target selector. Since a high-affinity clone typically gives a yield of ~1% in the affinity-selection process, that means that every clone of interest should be represented by at least ~100 TU (equivalent typically to ~2000 virions). This requirement is most difficult to meet in the first round of selection from the initial phage display library, in which all clones in the primary library—including the clones of interest—are about equally represented. For a library with 109 primary clones, for instance, the input should have at least 1011 TU (~2 × 1012 virions), and at least ten times that number if feasible. In contrast, in the inputs to the second and subsequent rounds of affinity selection (i.e., the amplified eluates from the previous rounds), the clones of interest are overrepresented and the total number of TUs can be reduced if necessary without compromising the goals of the project. There is no reason in most circumstances for the input phage to be purified in any way; indeed, we sometimes use the culture supernatant from eluate amplification directly as the input to the next round of affinity selection.

Internal enrichment control phage

Phage fd-cat is described in VECTORS.DOC. Its 7775-base genome is derived from fd-tet by replacing the tetracycline resistance determinant with the chloramphenicol acetyl transferase (cat) gene from plasmid vector pBR328. It has the same replication defect as fd-tet; its infectivity is roughly 5 times less on average (typically ~1%). These phage are propagated and titered (TUtiter.doc) the same way as fd-tet-derived phage, using chloramphenicol (34 µg/ml in both plates and liquid medium) in place of tetracycline.

Since fd-cat virions display no foreign peptides and can be titered independently of fd-tet-derived library phage, they can serve as an internal indicator of non-specific background yield during affinity selection. Thus fd-cat virions are mixed with the input phage (usually equal numbers of virions of each type) prior to each round of affinity selection. Then, by comparing the yield of library phage (tetracycline TU) with the yield of fd-cat control phage (chloramphenicol TU) in the eluates (step 13–17 below), the progress of enrichment at each round of affinity selection can be quantified. Because substantial numbers of fd-cat control virions are added to the input of each round of affinity selection, they should be propagated and purified on a large (e.g., 2-liter) scale (VirionPurification.DOC, without detergent treatment).

Eluates must be amplified before serving as inputs to subsequent rounds of affinity selection

If the eluate (output) from a round of affinity selection is to serve as the input to a subsequent round, it must first be amplified. There are two reasons for this. First, and most obviously, amplification is necessary to counter the low yield generally attained in affinity selection (typically only ~1% for a phage clone displaying a peptide with high affinity for the selector). Second, and less obviously, the elution process can cause subtle physical damage to the virions even if it doesn’t impair their infectivity substantially. Such damage can substantially increase non-specific background yield and thus slow the progress of enrichment for the desired selector-binding clones.

Amplification is achieved by infecting the eluate phage en masse into a large excess of fresh cells, propagating those cells in the presence of tetracycline (to select against uninfected cells), and partially purifying the secreted virions. If the eluate contains fd-cat internal control phage (conferring resistance to chloramphenicol; see the previous subsection) as well as library phage (conferring resistance to tetracycline), the former will be strongly selected against during propagation in the presence of tetracycline. There may be a few residual fd-cat phage in the amplified eluate because of the freeloader effect (see VECTORS.DOC), but such contaminants will be negligible compared to the fd-cat control phage that are purposely added prior to a subsequent round of affinity selection.

One-step selection

NOTE: When the amount of biotinylated selector added at step 4 below is enough to saturate the immobilized streptavidin (1–10 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 our libraries, this high yield is plausibly attributed to attachment of a single virion to two or more neighboring selector 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 selector 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.

1. Coat a 35-mm petri dish with 400 µl 10 µg/ml streptavidin in 0.1 M NaHCO3 for at least 1 hr at room temperature or overnight at 4ºC in a humid box (a sealed plastic box with a damp paper towel on the bottom).

2. Aspirate out the streptavidin solution and fill the dish to brimming with blocking solution; allow to sit with lid off for 2 hr at room temperature.

3. Pour the blocking solution back into its container (we continue to re-use it until it becomes cloudy). Wash five times with TBS/Tween from a squirt-bottle, emptying the dish by aspiration each time.

4. Add the desired amount of biotinylated selector (0.01–10 µg, the latter being recommended in the first round) in 400 µl TTDBA; allow the dish to react at least 2 hr at 4°.

5. Wash five times with TBS/Tween as in step 3 to remove unbound selector, and fill the dish with 400 µl of TTDBA. Add 4 µl 10 mM biotin and rock at room temperature for 10 min in order to block unoccupied biotin-binding sites on the immobilized streptavidinin.

6. Add the input phage (see “Input phage” in the introduction), premixed with fd-cat internal enrichment control phage if desired (see “Internal enrichment control phage” in the introduction; a small amount of the premix should be saved for titering at step 17 below), and rock the dish for 4 hr (usually at 4°, but sometimes at other temperatures). There is no need to remove excess free biotin: it won’t displace the bound biotinylated selector.

7. Wash the dish ten times with TBS/Tween as in step 3.

8. Elute bound phage from the dish with 400 µl of elution buffer for 10 min on a rocker; transfer the eluate to a microtube and neutralized by mixing it with 50 µl of 1 M Tris-HCl (pH 9.1). If the elution buffer is yellow as a result of having 0.1 mg/ml phenol red, neutralization will turn the color rosé. This is the unamplified eluate. If this is not the last round of selection, amplify the eluate as detailed in steps 13–22. If it is the last eluate, titer the unamplified eluate and propagate individual clones as detailed in steps 23–25.

Two-step selection

9. Equilibrate input phage (see “Input phage” in the Introduction), premixed with fd-cat internal enrichment control phage if desired (see “Internal enrichment control phage” in the introduction; a small amount of the premix should be saved for titering at step 17 below), overnight at 4º with the desired amount of biotinylated selector (usually less than 1 µg) in TTDBA (typically ~100 µl).

10. Meanwhile, coat a 35-mm dish with streptavidin as in steps 1–3 above and fill it with 400 µl TTDBA.

11. Add the reaction mixture (step 9) to the streptavidin-coated dish from the previous step. Rock for 10 min at room temperature to permit capture by immobilized streptavidin.

12. Wash and elute the dish as in steps 7–8 above.

NOTE: During the equilibrium step (step 9), selectors (assuming they are monovalent) bind phage reversibly according to solution-phase equilibrium kinetics. If there is little dissociation and reassociation during the subsequent 10-min capture step (step 11), the situation at the beginning of the capture step will largely determine the relative yields of different clones. If, at the other extreme, selectors 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 selector at high concentration. In practice, two-step selection gives considerably lower yields than one-step selection, even when reassociation is not suppressed.

Quantifying yield and amplifying eluates

NOTE: 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 (step 13) to allow the entire eluate to be amplified (see “Yield is all-important in the first round of selection” in the Introduction). Eluates from subsequent rounds, in which every clone is represented by many thousands or millions of phage particles, are used without concentration.

13. (For first-round eluates only) Concentrate the entire first-round eluate and wash it once with TBS on a Centricon 30-KDa ultrafilter (Amicon) by centrifuging at 5 Krpm in the Sorvall SS34 rotor (with thick-walled rubber adaptor) to gave a final volume of 100 µl.

14. In a 1.5-ml Ep tube mix 100 µl of eluate (the entire eluate if from the first round) and 100 µl of starved K91BluKan (= K91BK) cells (section A of TUtiter.doc) or K91BK terrific broth culture (section B of TUtiter.doc); incubate 10–30 min at room temperature.

15. Pipette the infected cells into a 250-ml culture flask containing 40 ml NZY with 0.2 µg/ml tetracycline[1]; shake 30–60 min at 37°.

16. Spread 200-µl portions of appropriate serial dilutions of the culture (diluent = NZY) on NZY plates containing 40 µg/ml tetracycline and 100 µg/ml kanamycin to quantify the output of the affinity selection. If the input was premixed with fd-cat internal control phage (see step 6 or 9), also spread 200-µl portions of appropriate serial dilutions on NZY plates containing 34 µg/ml chloramphenicol and 100 µg/ml kanamycin to quantify the background output.

NOTE: Assuming the input to the affinity selection was ~1011 TU, as recommended, dilutions in the range of 10-1 to 10-4 will cover yields in the range of 3 × 10-5% (typical non-specific background) to 1% (the highest yields we ordinarily observe). For the first round, the 10-1 and 10-2 dilutions will almost always suffice. The yield of the added fd-cat internal enrichment control phage (if included) should remain at background levels at all rounds of affinity selection.

17. At the same time, titer a suitable serial dilution of the input phage (or of the input phage/fd-cat premix if relevant) (diluent = TBS/gelatin; dilution should aim at ~105 TU/ml, equivalent to ~2 × 106 physical particles/ml) by the ordinary analytical titering method (section C of TUtiter.doc) on the same starved cells or terrific broth culture as were used in step 14. If fd-tet internal enrichment control phage were included at step 6 or 9, titer for chloramphenicol TU as well as tetracycline TU. The yield of affinity selection can be calculated by dividing the total number of output TU by the total number of input TU.

NOTE: A suitable dilution of an initial library with a physical particle concentration of, say, 2 × 1014 virions/ml (~1013 TU/ml) is 10-8; suitable dilutions of an amplified eluate with a physical particle concentration of ~5 × 1013 virions/ml are 10-7 and 10-8.

18. Meanwhile, add 40 µl of 20-mg/ml tetracycline to the 40-ml culture step 15 to bring the total antibiotic concentration to 20 µg/ml; continue shaking overnight at 37º. Cells harboring fd-cat internal enrichment control phage, if present, will be killed by the tetracycline, and library phage should therefore greatly predominate in the culture after overnight growth in that antibiotic (see “Internal enrichment control phage” in the introduction).

19. Pour the culture into a 50-ml tube and clear it of cells by two 10-min centrifugations at 5 and 8 Krpm in a FiberLite rotor[2] at 4º. Pour the doubly-cleared supernatant into a fresh 50-ml tube and note volume (usually ~35 ml).

NOTE: As noted above (see Input phage in the Introduction), there’s usually no need to purify the amplified phage in any way in preparation for subsequent rounds of affinity selection. Nevertheless, we generally purify the virions by two successive PEG precipitations (next three steps) in the hope that the phage are less likely to lose affinity to contaminating proteases upon prolonged storage.

20. To the doubly-cleared culture supernatant previous step add 0.15 vol (~5.25 ml) PEG/NaCl and mix by many inversions; allow phage to precipitate overnight in the refrigerator.

21. Collect the precipitated phage by centrifuging at 12 Krpm 15 min at 4º in the FiberLite rotor; RRR; dissolve pellet in 1 ml TBS and transfer the solution to a 1.5-ml Ep tube; microfuge 1 min at top speed to clear insoluble material; carefully transfer the supernatant to a second 1.5-ml Ep tube.

22. Add 150 µl PEG/NaCl; vortex to mix; refrigerate at least 1 hr; microfuge 5 min at top speed; RRR; dissolve pellet in 400 µl TBS; microfuge 1 min at top speed to clear undissolved material; transfer supernatant to a 500-µl Ep tube; store in refrigerator. This is the amplified eluate; the physical particle concentration should be ~5 × 1013 virions/ml[3], regardless of the titer in the unamplified eluate; the titer is ~0.5–5 × 1012 TU/ml. Usually we use 100 µl of this amplified eluate for the next round of affinity selection.

NOTE: If the amplified eluate is to be stored for a long time, it’s a good idea to add NaN3 to a final concentration of 0.02–0.05% to inhibit microbial growth.

Quantifying yield and propagating clones from the final eluate

NOTE: 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 (the unamplified eluate, step ). 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. If fd-cat internal enrichment control phage were pre-mixed with input phage at step 6 or 9, titer that mixture and the unamplified final eluate for both tetracycline and chloramphenicol TU at steps 23–24; propagate only the tetracycline-resistant clones at step 25.

23. Using TBS/gelatin as diluent, make suitable serial dilutions of both the input (see note under step 17) and the unamplified final eluate (step 8 or 12).

NOTE: Assuming an input of 1011 TU, suitable dilutions of the output (unamplified eluate; see step 8; volume 450 µl) are 10-1 to 10-5. This will cover yields up to ~5%.

24. Titer these dilutions on starved cells (section B of TUtiter.doc) by the analytical titering procedure (section C of TUtiter.doc). If fd-tet internal enrichment control phage were included at step 6 or 9, titer both the input/fd-cat premix and the unamplified eluate for chloramphenicol TU as well as tetracycline TU. The yield of affinity selection can be calculated by dividing the total number of output TU by the total number of input TU.

25. Propagate the desired number of well separated output tetracycline-resistant colonies from the output titering for individual characterization. In most cases, these clones are propagated and processed on the 7-ml scale and crude double-stranded RF prepared as in RFminiprep2.doc. Meanwhile, the culture supernatants from the 7-ml propagations can be processed for partially purified virions as described in SmallScaleVirions.doc; these virions can be used, for example, for binding assays as described in micropan.doc and ELISA.doc.

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[1] This concentration of tetracycline is sub-inhibitory but sufficient to derepress the inducible tetA tetracycline resistance gene. It does not interfere with titering of fd-cat chloramphenicol TU.

[2] These excellent rotors allow you to spin standard disposable 50-ml conical screw-cap centrifuge tubes at 15,000 rpm (not relevant here, though). The pellet collects at the angle rather than the tip, which makes it particularly easy to free of residual supernatant. If you don’t have this rotor, you can use OakRidge tubes and the Sorvall SS34 rotor (or equivalent Beckman rotor) instead.

[3] It’s a good idea, if convenient, to confirm this estimate empirically by scanning a 1/20 dilution from 240 to 320 nm and calculating the physical particle concentration as described in AbsorptionSpectrum.doc.

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