Notes
Affinity Selection of Peptide Binders with Magnetic Beads via Organic Phase Separation (MOPS)
2015 Volume 38 Issue 11 Pages 1822-1826
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2015 Volume 38 Issue 11 Pages 1822-1826
We describe a new method for affinity selection of peptide binders for soluble protein targets using magnetic beads via organic phase separation (MOPS) from a phage display library. As a model target molecule, a mouse monoclonal antibody against human integrin α9β1 (Y9A2) immobilized onto protein G magnetic beads was incubated with a 15-mer or 20-mer random peptide phage-display library. The suspensions containing the phage-magnetic beads conjugates were then transferred onto the organic phase and centrifuged in order to recover the Y9A2 bound phage immobilized on the protein G magnetic beads in the lower organic phase. After three rounds of biopanning, we were able to isolate specific phage clones that could not be obtained by the conventional approach. Furthermore, this new approach was found to be highly effective for isolating phage-binders for Fc-fusion constructs; indeed, enrichment of specific phage-binders was observed after only the first panning cycle. Thus, MOPS can improve the selection of specific phage-binders for soluble protein targets mainly due to the removal of non-specific binders.
Phage display has been used extensively to generate peptide ligands that possess binding affinity towards a variety of targets. Traditionally, several rounds of affinity selection (formally known as “biopanning”) that rely on washing steps are performed to enrich high affinity binders. Conventional biopanning, however, sometimes leads to the isolation of false positive clones mainly due to their unspecific binding to components such as the solid phase, target-capturing molecules, blocking agents, etc.1) Subtracting selection, where the background yield is reduced by preincubating the input phage with the solid phase coated with a capturing and blocking agent prior to incubation with target-coated solid phase,2) and specific elution with known target ligands seem to be effective in removing target-unrelated phage.3) Unfortunately, it may still be difficult to efficiently reduce the non-specific phage-binders.
Organic phase separation techniques such as biopanning and rapid analysis of selective interactive ligands (BRASIL)4) and isolation of antigen-antibody complexes through organic solvent (ICOS)5,6) have been successfully applied to the screening of cell-surface-peptide binders or cell-surface-binding monoclonal antibodies from a phage display library. These methods allow effective separation of phage-target complexes from unbound phage by centrifugation through organic solvent based on the fact that free phage are retained in the upper aqueous phase, while phage-target complexes are pelleted in the organic phase. In addition to the specificities of isolated clones, their higher affinities have also been reported by ICOS.5)
In this study, we applied the concept of organic phase separation to affinity selection of phage-binders against soluble protein targets through the use of magnetic beads onto which a target protein was immobilized (designated magnetic beads via organic phase separation (MOPS)). MOPS was found to be a valid and efficient approach to select specific phage-binders that could not be obtained by conventional biopanning.
The fUSE55 vector and an associated Escherichia coli host strain (K91BK) were kindly provided by Dr. George P. Smith (University of Missouri, Columbia, MO, U.S.A.). Phage-display peptide libraries with 15 or 20-mer random sequences fused to pIII were constructed as described previously.2) Fifteen and 20-mer random peptide libraries contained 4.8×108 and 2.2×108 individual clones, respectively. Detailed protocols for the construction of phage-display random peptide libraries are available from Dr. George P. Smith’s website: http://www.biosci.missouri.edu/smithGp/PhageDisplayWebsite/PhageDisplayWebsiteIndex.html.
Phage concentration was determined by measuring the absorbance value of phage solution at 269 and 320 nm (see Dr. George P. Smith’s website). Phage concentration was calculated by the following formula:
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We chose Y9A2 (sc-59969, Santa Cruz Biotechnology, Inc., Santa Cruz, CA, U.S.A.), a mouse monoclonal antibody against human integrin α9β1,7) as a model target molecule. Biopanning selection based on binding affinity of 15-mer peptide binders to immobilized Y9A2 in a 96-well microtiter plate (Maxisorp, Nunc, Thermo Fisher Scientific Inc., Waltham, MA, U.S.A.) was performed as described previously.8) Briefly, a well was coated overnight at 4°C with 2 µg of Y9A2 in 100 µL of phosphate-buffered saline (PBS) and blocked with 1% (w/v) bovine serum albumin (BSA) and 0.05% (v/v) Tween 20 in PBS (dilution buffer) for 30 min at room temperature. A 15-mer random phage-display library (approximately 1012 virions) in 100 µL of dilution buffer was incubated in the Y9A2-coated wells for 2 h at room temperature. Unbound phage were removed by washing four times with washing buffer (PBS containing 0.05% (v/v) Tween 20) and bound phage were eluted with 0.1 M glycine–HCl (pH 2.5) containing 1 µg/mL BSA. Eluted phage were neutralized and then amplified using E. coli K91BK. Biopanning was repeated for three rounds.
Screening of Y9A2 Binding Phage via Organic-Aqueous Environment (MOPS Method)We tried two procedures for biopanning. In the first procedure, known as the solution binding approach (Screening A), the complex of pre-reacting phage with Y9A2 in dilution buffer was captured with protein G sepharose magnetic beads. In the second procedure, phage were screened with pre-immobilized Y9A2 onto protein G sepharose magnetic beads (Screening B). All 1.5-mL centrifuge tubes used for biopanning were blocked with dilution buffer (PBS containing 1% (w/v) BSA and 0.05% (v/v) Tween 20) prior to affinity selection. All incubation steps were performed at room temperature in a 1.5-mL centrifuge tube with rotation at 700 rpm on a microplate mixer (CM-1000, Tokyo Rikakikai Co., Tokyo, Japan). The washing and elution steps were conducted using magnetic separation.
Screening AVirions (1×1012) of a 15-mer or 20-mer random peptide library and 1 µg of Y9A2 in 20 µL of dilution buffer were incubated in a 1.5-mL centrifuge tube. After 2 h, 5 µL of PBS-equilibrated protein G sepharose magnetic beads (Protein G Mag sepharose, GE Healthcare UK Ltd., Buckinghamshire, U.K.) were added for affinity capture of the phage-Y9A2 complexes. After an incubation time of 30 min, the tube was placed on a magnet and the supernatant was removed. The magnetic beads were washed four times with 500 µL of PBS-T (PBS containing 0.05% (v/v) Tween 20) and resuspended in 500 µL of PBS-T. The magnetic beads suspension was overlaid onto 500 µL of organic solution (dibutyl phthalate–cyclohexane (9 : 1, v/v) in a 1.5-mL centrifuge tube. The tube was centrifuged at 5000 rpm (2400×g) for 2 min at 4°C. After removal of the supernatant, the magnetic beads were suspended in 500 µL of PBS-T and this suspension was overlaid again onto 500 µL of the organic solution in a new 1.5-mL centrifuge tube. This organic phase separation was repeated thrice. After the organic phase separation, the magnetic beads were washed four times with 1 mL of PBS-T. Bound phage were eluted with 20 µL of 0.1 M glycine–HCl (pH 2.5) containing 1 µg/mL BSA (5 min incubation). Phage eluate was neutralized in a new tube with 1 µL of 1M Tris–HCl (pH 9.5) and amplified in E. coli K91BK. Three rounds of biopanning were performed.
Screening BOne microgram of Y9A2 was pre-reacted with 5 µL of PBS-equilibrated protein G sepharose magnetic beads in 20 µL of dilution buffer for 30 min. The magnetic beads were washed with 50 µL of PBS-T. A 15-mer or 20-mer random peptide library (1×1012 virions) was subsequently added and incubated in 20 µL of dilution buffer for 2 h. Next, the magnetic beads were washed four times with 500 µL of PBS-T and resuspended in 500 µL of PBS-T. The procedures for the organic phase separation and the elution of bound phage were the same as those followed for screening A. Biopanning was repeated for three rounds.
Screening of Human Granulocyte-Colony Stimulating Factor Receptor (G-CSF) Receptor Binding Phage via Organic-Aqueous Environment (Using MOPS)Fc region of mouse immunoglobuline G1 (IgG1) and the Human G-CSF receptor-Fc fusion composed of an immunoglobulin-like [Ig] domain, and a cytokine receptor homologous [CRH] region of G-CSF receptor were kindly provided by Dr. Ryota Kuroki (Japan Atomic Energy Research Institute, Ibaraki, Japan).9) Screening B of MOPS was used to select phage-binders for G-CSF receptor-Fc. The MOPS procedures were the same as those followed for Y9A2, except that 5 µg of G-CSF receptor-Fc were used instead of 1 µg.
Phage Enzyme-Linked Immunosorbent Assay (ELISA)Phage purified with polyethylene glycol precipitation from each round of biopanning were subjected to an analysis by a phage enzyme-linked immunosorbent assay (phage ELISA) as described previously.2) Each well of a 96-well microtiter plate (Maxisorp, Nunc, Thermo Fisher Scientific Inc., Waltham, MA, U.S.A.) was coated with 100 µL of PBS containing 1.0 µg/mL Y9A2, G-CSF receptor-Fc or mouse IgG (Sigma-Aldrich, Saint Louis, MO, U.S.A.) at 4°C overnight. The wells were blocked with PBS-T containing 1% (w/v) BSA (dilution buffer) for 30 min at room temperature. Then, 1010 virions/100 µL of dilution buffer were incubated in each well for 1 h at room temperature and then washed three times with PBS-T. Hundred microliters of horseradish peroxidase (HRP)-labeled anti-M13 monoclonal antibody (GE Healthcare UK Ltd., Buckinghamshire, U.K.) in dilution buffer at a dilution of 1 : 5000 (v/v) were incubated for 1 h at room temperature in each well. After the plate was washed three times, 100 µL of substrate solution (SureBlue TMB Microwell peroxidase substrate, KPL, Gaithersburg, MD, U.S.A.) was pipetted into each well and incubated for 5 min at room temperature. The reaction was stopped by adding 100 µL of 1 N HCl and absorbance at 450 nm was measured in a microplate reader.
Organic phase separation is reported to be an efficient approach for separating cell-bound phage from free phage by centrifugation during biopanning (BRASIL4) and ICOS5,6)). Here, we extended this concept of organic phase separation onto the biopanning of a random peptide phage display library against soluble protein targets instead of those expressed at the cell surface. Y9A2, a mouse IgG1 antibody against human integrin α9β1,7) was chosen as a model of a soluble protein target. Based on the ICOS method, we developed a screening in which protein G magnetic beads are used instead of cells through organic phase separation (designated MOPS). After the incubation of a random peptide phage display library with Y9A2, protein G magnetic beads on which the phage-Y9A2 complexes are affinity-captured are overlaid onto the organic solution, followed by removal of unbound phage through centrifugation. We repeated this organic phase separation three times for obtaining isolated phage with higher specificities. After the organic phase separation, bound phage were eluted by lowering the pH (Fig. 1). Biopanning was repeated thrice.
To remove non-specific clones, organic phase separation was repeated three times.
Despite intensive screenings of random peptide phage display libraries, Y9A2 binding phage have not yet been obtained by conventional biopanning that relies on washing steps, which makes Y9A2 an interesting soluble protein target. Figure 2 shows the typical phage ELISA results by which the isolated phage from each round of four independent conventional biopannings were analyzed (see Materials and Methods). By the conventional screening method, no phage specific to Y9A2 could be obtained due to high enrichment of background-bound phage in most screenings. Thus, distinguishing the specific phage from background-bound phage is an important step towards a successful selection of phage display libraries. Unfortunately, use of protein G sepharose beads on which Y9A2 was immobilized did not lead to a successful selection of specific phage-binders (data not shown). Next, we screened phage-displayed random peptide libraries with Y9A2 by MOPS. Two biopanning procedures were carried out: one presented Y9A2 in the liquid phase (screening A), the other presented it pre-immobilized onto protein G sepharose magnetic beads (screening B). Except for one instance (20 mer-B in Fig. 2B), specific Y9A2 binding phage were obtained by both screenings A and B. We confirmed that Y9A2 (mouse IgG1) binding phage from the 3rd round (15 mer-A, B and 20 mer-A in Fig. 2B) did not cross-react with mouse IgG (polyclonal mouse IgG behaves as if it were an isotype control because a large portion of polyclonal IgG is IgG1), suggesting that the phage isolated in this manner are specific (data not shown). It is worth noting that phage bound to the solid phase enriched in the 1st round of 20 mer-B screening can be efficiently removed in the 2nd and 3rd round of MOPS, while the background-bound phage panned by the conventional approach tended to increase with repetitive rounds (see 15 mer-3 and -4 in Fig. 2A). Therefore, MOPS is an efficient approach to detect the specific peptide binders that could be missed by conventional phage screening techniques.
Phage purified with polyethylene glycol precipitation from each round of biopanning were analyzed by phage ELISA. Gray bars: wells coated with Y9A2; white bars: wells coated with bovine serum albumin (BSA) only. NC15 is a target-unrelated phage used as a negative control. All of the data were calculated from three wells (mean±S.D.). A: Affinity selection of Y9A2 binding phage in a 96-well microtiter plate (conventional biopanning). A 15-mer phage-displayed random peptide library was screened in Y9A2-coated wells for three rounds (four independent screenings were performed simultaneously). Enrichment of background-bound phage was observed in most screenings. B: Affinity selection of Y9A2 binding phage via organic phase separation (MOPS). Fifteen and 20-mer phage-displayed random peptide libraries were screened with Y9A2 via organic phase separation for three rounds. Enrichment of Y9A2-bound phage was observed in most screenings. 15 mer-A, 20 mer-A: biopanning by screening A; 15 mer-B, 20 mer-B: biopanning by screening B.
To further confirm the applicability of the MOPS method, we applied this new approach to the affinity selection of phage-binders against the mouse IgG1 Fc-fusion construct of human G-CSF receptor.9) MOPS was found to be a very efficient approach. Indeed, MOPS allowed for the selection of specific phage-binders after only the 1st round in four independent screenings (Fig. 3), while no phage-binder was obtained by conventional biopanning (data not shown). A well-known concern is that the amplification step during the biopanning leads to the loss of sequence diversity and real binding clones as well as to the increase of propagation-related target-unrelated phage.1,10) Reducing the number of amplification steps in biopanning is the only way to address these issues.1) From this viewpoint, MOPS may be a good way for selecting peptide-binders.
Human G-CSF receptor-mouse IgG1 Fc fusion was used to screen phage-display random peptide libraries by screening B of MOPS. MOPS is an effective approach for isolating peptide binders specific to the Fc-fusion because target-bound phage can be obtained from the 1st round on. The data represent means from duplicate wells. NC15 is a target-unrelated phage used as a negative control. No cross-reactivity of selected phage was observed with mouse IgG.
In MOPS, protein G magnetic beads are effectively used to achieve affinity capture of mouse IgG and Fc fusion, because they are easy to separate from the liquid phase with a magnet. Compared to a 96-well microtiter plate, magnetic beads possess increased surface area, leading to a more efficient phage panning.11,12) We combine the advantage of magnetic beads with organic phase separation to design affinity selection of peptide binders with high specificity. Other commercially available magnetic beads, such as poly-His, glutathione S-transferase and maltose-binding protein, are also applicable to MOPS; although the affinity of each tagged-protein with its respective magnetic beads in the organic solvent should be verified. Otherwise, non-tagged proteins can be covalently coupled to the N-hydroxysuccinimide (NHS) functional groups on magnetic beads via their primary amino groups.
In conclusion, MOPS, in which the organic phase separation is performed, is an efficient method to identify the phage-binders against soluble protein targets from a random peptide phage display library. Owing to low background, MOPS can lead to the improved selection of specific phage-binders that could not obtained by conventional biopanning.
We thank Dr. Yasushi Akahori (Fujita Health University) for having helpful discussions with us about the organic phase separation. We are also grateful to Dr. Ryota Kuroki (Japan Atomic Energy Research Institute) for kindly providing the human G-CSF receptor-Fc fusion.
The authors declare no conflict of interest.
