Chromato-panning: an efficient new mode of identifying suitable ligands from phage display libraries
© Noppe et al; licensee BioMed Central Ltd. 2009
Received: 25 August 2008
Accepted: 17 March 2009
Published: 17 March 2009
Phage Display technology is a well established technique for high throughput screening of affinity ligands. Here we describe a new compact chromato-panning procedure for selection of suitable binders from a phage peptide display library.
Both phages and E. coli cells pass non-hindered through the interconnected pores of macroporous gel, so called cryogel. After coupling a ligand to a monolithic cryogel column, the phage library was applied on the column and non-bound phages were washed out. The selection of strong phage-binders was achieved already after the first panning cycle due to the efficient separation of phage-binders from phage-non-binders in chromatographic mode rather than in batch mode as in traditional biopanning procedures. E. coli cells were applied on the column for infection with the specifically bound phages.
Chromato-panning allows combining several steps of the panning procedure resulting in 4–8 fold decrease of total time needed for phage selection.
Since the introduction of the phage display peptide libraries in 1985 , when a vector could directly display a small foreign peptide in one of the coating proteins of the filamentous M13 phage particle, the greatest task has been to find suitable ligands. Because of the need for repeated panning rounds to isolate high-affinity ligands, the whole panning procedure is rather laborious and time consuming. Therefore, a shorter, more efficient procedure, with fewer steps and fewer panning cycles would be desirable.
Phage display is suitable not only for peptides, but also for small proteins and protein fragments (e.g. antibody fragments), which can be expressed on the phage surface proteins . Several linear [3–6] and constrained cyclic [7–10] phage peptide libraries have been sucessfully used to screen for affinity ligands against a variety of targets, all of which were based on use of the laborious, repetitive biopanning procedure. The technique of phage display and its applications is more extensively discussed in some recent review papers. C. Adda et al.  and J. Kehoe et al. review the technique of phage display in all its aspects. M. Paschke ; M. Szardenings ; M. Arap ; C. Mersich et al.  and V. Petrenko  present extensive reviews on the present state and applications of the phage display technology.
(i) be macroporous, with a pore size no less than 10 μm, as the phages themselves are about 1 μm long (larger pore sizes, up to 100 μm, are desirable to allow the possibility of direct infection of the cells by the phages bound to the affinity matrix);
(ii) have highly interconnected pores, allowing convectional rather than diffusional mass transport of phages and presumably cells inside the pores;
(iii) be hydrophilic with minimal non-specific binding of phages or cells and hence no need for post-coating;
(iv) contain active groups for the immobilization of the ligand.
We have recently presented a material with the required combination of properties for chromatographic separation of microbial and mammalian cells [17–20]. Macroporous hydrogels, or cryogels, are produced using polymerization in semi-frozen conditions, where ice crystals perform the role of the porogen, and polymerization proceeds in the non-frozen liquid phase . The properties of cryogels and the architecture of interconnected macropores are presented in detail in recent publications by Plieva et al. [22–24]. In this communication we present a new chromato-panning procedure based on using a cryogel as a matrix for ligand immobilization.
Results and discussion
Statistical analysis data
To further optimize the column panning procedure, on-column infection of E. coli cells was investigated. The macropores in the cryogel are large enough to allow E. coli cells to pass freely through the plain cryogel without blocking the pores . However, on the HuLF cryogel we observed binding of cells that could be eluted from the cryogel with NaCl, in accordance with previous observations that E. coli cells bound to an anion-exchange cryogel column were eluted by NaCl . As HuLF is positively charged at pH ~7.0, the HuLF-cryogel may act as an "anion-exchanger", which could explain our observation. NaCl (1 M) eluted all cells from the column. By immediately diluting the 1 M NaCl eluate in Luria broth (LB) medium, the final NaCl concentration of the eluted cell suspension was reduced to approx. 0.4 M NaCl. This NaCl concentration had no negative effect on the viability of the cells. Under these conditions we observed a similar growth rate on agar plates to that of cells that had not come into contact with high salt concentrations.
To further confirm the on-column infection of the E. coli cells we measured the amplification rate of the E. coli cells after infection on the column. E. coli cells that were not in contact with phages (blank sample) did not grow on agar plates containing tetracycline. E. coli cells that were infected by phages on the column survived and grew on the agar plates containing tetracycline (Figure 3c). After the cells were infected and eluted from the column, the cell suspension was centrifuged and the pellet re-suspended in LB buffer. At this point, the optical density at 600 nm (OD600) was measured. After plating, overnight incubation at 37°C and harvesting the cells in LB medium, OD600 nm was measured again. A ~950 fold increase in OD600 was observed, which indicates that the cells were effectively infected on the HuLF cryogel column. The new panning procedure is independent of the ligand or phage library used. We used different phage peptide libraries and two different proteins as the ligand. The results presented above are based on results obtained with HuLF as ligand. Similar results were obtained in an experiment with α-chymotrypsin as the ligand and the L6 phage peptide library (data not shown).
Comparative time frame of chromato-panning protocol and tube panning protocol
Round 1 (h)
Round 1 (h)
On column panning
On column infection
No additional panning rounds are needed
2 additional panning rounds are performed
Phage incubation R2/3
Comparative time frames of reported panning protocols
Microtiter plate 
Microtiter plate 
Round 1 (h)
Round 1 (h)
Round 1 (h)
Cell growth tubes
2 additional panning rounds
2 additional panning rounds
3 additional panning rounds
The total time is considerably shorter when using a column procedure (Table 2). Advantages of the column procedure are: no target coating is needed for each round as the column is re-usable, no post-coating is needed, the contact time between the phage-containing solution and the column is shorter and, finally, a milder elution medium can be used. The first column panning round takes about 1/3 of the time required for the first round in a tube panning procedure. If three rounds were needed a reduction of approximately 50% in time is obtained. Only 1 round was necessary to obtain high-affinity binders in the column panning procedure, compared to 3 rounds in the tube panning procedure. In this case column panning takes only approximately 1/6 of the time needed for a tube panning.
The use of macroporous cryogels in a chromato-panning procedure has various advantages. The large interconnecting pores of the cryogel allow (i) free passage of bacteriophages during the panning step without clogging the column, and (ii) free passage of E. coli cells so that infection can take place on-column. (iii) A significant time saving can be achieved as only one column panning round is needed to select high-affinity phage clones. (iv) A milder elution solvent can be used to elute phages and E. coli cells, without denaturing the bound ligand on the column or having negative effects on the viability of the cells. At present we provided "proof-of-principle", it is clear that our procedure as well as the data need to be more extensively validated in the future before the method can be broadly used as an alternative for the classical biopanning and as a new platform for rapid screening of ligands.
Preparation of cryogel monoliths
The epoxy-activated macroporous cryogels were produced and provided by Protista Biotechnology AB (Lund, Sweden) according to published protocols .
Tube biopanning procedure
A solution of human lactoferrin (HuLF) at 10 μg/ml in phosphate-buffered saline (PBS) was coated for 2 hours at room temperature (RT) on sterile Nunc immunotubes (Nunc A/S) placed on a test-tube rotator at 10 rpm, followed by standing incubation overnight at 4°C. (Figure 1a). After discarding the coating solution the tubes were washed with PBS containing 0.2% Tween-20 (TPBS). Post-coating was performed with a 4% (wt/vol) solution of milk powder in PBS for at least 2 hours at RT. After washing with TPBS, 2 × 1012 phages from a phage-display peptide library were added in PBS to the tube and incubated overnight at 4°C on a test-tube rotator at 10 rpm. After careful washing with TPBS, the bound phages were eluted with 0.1 M glycine, pH 2.0, on a test-tube rotator for 15 min at RT. The eluate was neutralized by adding 1 M Tris pH 8.0 and kept on ice until infection. An overnight E. coli TG-1 (Stratagene) cell culture was diluted 100 times and grown at 37°C on a shaker until the OD600 was ~0.500. To 650 μl eluted phages, 10 ml of the E. coli TG-1 cell culture was added and incubated at 37°C for phage infection. After incubation, the cell suspension was centrifuged and the cell pellet was re-suspended in sterile Luria broth (LB) medium (Invitrogen). The cell suspension was subsequently plated on Luria agar plates containing 20 μg/ml tetracycline-hydrochloride, and incubated overnight at 37°C.
The cells were recovered in sterile LB medium and the cell suspension was centrifuged for 20 min at 7600 × g at 4°C. To the clear supernatant, 1/3 to 1/5 volume of 20% PEG-6000/2.5 M NaCl was added, mixed and incubated on ice for 90 min for phage precipitation. Phages were recovered by centrifugation at 2000 × g for 20 min. The pellet was re-suspended in sterile distilled water and precipitated again with the PEG/NaCl solution. After incubation and centrifugation the phage pellet was finally re-suspended in PBS. The phage concentration was measured as the absorbance at 260 nm (A260) (1A unit = 2.2 × 1011 phages.ml-1).
In order to obtain phages specific to the ligand, three panning rounds were performed. In the consecutive panning rounds, 1 × 1011 phages of the previous panning round was used for incubation in the coated immunotube. The incubation time was reduced to 90 min on a test-tube rotator followed by standing incubation for 60 min. All ensuing steps were similar to those described for the first panning round. Finally, the phages were stored in PBS buffer – 5% (wt/vol) glycerol at -20°C until further use. All buffers were filtered through a 0.22 μm filter and autoclaved before use.
Ligand coupling to the monolithic cryogel
Two dried epoxy-activated monolithic cryogels were inserted, one on top of the other, in a chromatographic column. The monolithic cryogels were swollen in sterile water and the upper adaptor was lowered onto the gel surface (9 ml bed volume). The column was washed with sterile water and equilibrated with 0.1 M carbonate buffer, pH 9.5, at a flow rate of 1 ml/min. A solution of HuLF in 0.1 M carbonate buffer, pH 9.5, was re-circulated over the column for 16 hours at a flow rate of 0.5 ml/min at 4°C. After coupling, the column was washed with 0.1 M carbonate buffer, pH 9.5, until all non-bound HuLF had been removed, followed by re-circulation of 25 ml 0.1 M ethanolamine in 0.1 M carbonate buffer, pH 9.5, for 3 hours at a flow rate of 1 ml/min to block the remaining active epoxy groups. Finally, the column was washed with sterile water and PBS, and stored at 4°C until further use.
The HuLF-cryogel column (Figure 1b) was washed with sterile PBS. Two ml of solution containing 1 × 1012 phages of a phage-displayed peptide library was loaded onto the column at 0.5 ml.min-1, followed by washing with 30 ml PBS to remove unbound phages. The bound phage fraction was eluted, 1 ml fractions were collected and stored at -20°C until further use. Two different elution media were investigated: 0.1 M glycine, pH 2.0 (used in the tube panning procedure), and 1 M NaCl. After elution the column was washed with 2 M NaCl and re-equilibrated with PBS buffer. The eluted phages were amplified by infecting E. coli TG-1 cells as described above. Three panning rounds were performed using the same HuLF cryogel column.
On-column cell infection
The chromato-panning procedure was very similar to that described above. Instead of eluting the phages from the column, E. coli TG-1 cells were loaded onto the column for infection and phage amplification (Figure 1c). The phage-displayed peptide library was loaded onto the column, which was then washed with PBS to remove unbound phages. The column was then incubated at 37°C. An overnight E. coli TG-1 cell culture was diluted 100 times and grown at 37°C on a shaker until OD600 reached ~0.5. One column volume, approx. 8–9 ml cell suspension, was loaded onto the column and the flow was stopped. The cells were incubated on the column for 30 min at 37°C. The cells were then eluted from the column by washing with 20 ml LB medium containing 1 M NaCl. The eluate was collected in 20 ml LB medium to dilute the NaCl in order to maintain the viability of the E. coli cells. The cell suspension was immediately centrifuged at 750 × g for 10 min. The pellet was re-suspended in LB medium and plated on Luria agar plates containing 20 μg/ml tetracycline-hydrochloride, and incubated overnight at 37°C. Phage recovery was similar to that described above. A second round of chromato-panning and on-column infection was performed in a similar way. In the second round the phage concentration was reduced to 5 × 1010 phages.ml-1 (2 ml applied to the column). All buffers used had been filtered through a 0.22 μm filter and autoclaved. All procedures were performed at room temperature, except for the on-column infection and cell/phage elution which were performed at 37°C.
Human lactoferrin ELISA
The wells of a Microlon ELISA plate (Greiner Bio-One) were coated with 10 μg/ml of HuLF in PBS buffer and left overnight at 4°C. After washing with TPBS, the wells were post-coated with a 4% (wt/vol) solution of milk powder in PBS for at least 2 hours at RT. After washing, a 2-fold serial dilution of phages starting at 1 × 1011 phages/ml in 0.4% (wt/vol) milk powder in PBS was added to the wells and incubated for 2 hours at RT. After extensive washing with TPBS, HRP/anti-M13 monoclonal conjugate (GE Healthcare) (1:2500 in 0.4% (wt/vol) milk powder in PBS) was added and incubated for 1 hour at RT. After extensive washing, o-phenylenediamine hydrochloride and hydrogen peroxide solution was added. The reaction was stopped by adding 4 M sulfuric acid and the absorbance was measured at 490 nm using a BIO-Tek ELISA reader.
The wells of a Microlon ELISA plate were coated with 100 μl of 5 × 1010 phages.ml-1 in PBS buffer overnight at 4°C. After washing with TPBS, the wells were post-coated with a 4% (wt/vol) solution of milk powder in PBS for at least 2 hours at RT. After washing, a 2-fold serial dilution HuLF at 10 μg/ml in 0.4% (wt/vol) milk powder in PBS was added to the wells and incubated for 2 hours at RT. After extensive washing with TPBS, HRP/Goat anti-HuLF conjugate (Nordic) (1:5000 in 0.4% (wt/vol) milk powder in PBS) was added and incubated for 1 hour at RT. After extensive washing, o-phenylenediamine hydrochloride and hydrogen peroxide solution was added. The reaction was stopped by adding 4 M sulfuric acid and the absorbance was measured at 490 nm using a BIO-Tek ELISA reader.
The data (absorbance versus phages) for the three libraries (C6, L6 and L15) were fitted with a simple sigmoidal dose-response model, starting from Abs = 0 and ending at Abs = Top: Abs = Top/[1 + 10(logEC50 - X)], with X the log-transformed concentration of phages/ml (GraphPad Prism 5.0). The fit parameter logEC50 corresponds to the concentration of phages at half the saturation level. The fit parameter Top, along with its 95% Confidence Interval (CI), is used to represent the saturation level of the measured absorbance (for X → ∞). Statistical difference between saturation levels for tube panning after round 1 (R1) versus chromato-panning R1 for each library was evaluated based on non-overlapping 95% CI's (corresponding to p < 0.05). The good quality of the fits can be judged from the R2-value (explained variance).
An overall judgment of statistical difference for the three libraries was based on a paired t-test, comparing tube panning R1 with chromato-panning R1.
This work was financially supported by Protista Biotechnology AB (Lund, Sweden). W.N. expresses his gratitude for the fellowship provided by the Swedish Institute. Dr. S. Deroo (Laboratory of Retrovirology, Luxembourg) is acknowledged for the amplification of the L6 and L15 phage peptide libraries.
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