Journal of Immunological Methods 405 (2014) 121–129

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Research paper

Mimtags: The use of phage display technology to produce novel protein-specific probes Nayyar Ahmed a,b, Pathum Dhanapala a,b, Nadia Sadli a,b, Colin J. Barrow b, Cenk Suphioglu a,b,⁎ a b

NeuroAllergy Research Laboratory (NARL), Faculty of Science, Engineering and Built Environment, Deakin University, 75 Pigdons Road, Geelong, Victoria 3216, Australia School of Life and Environmental Sciences, Faculty of Science, Engineering and Built Environment, Deakin University, 75 Pigdons Road, Geelong, Victoria 3216 Australia

a r t i c l e

i n f o

Article history: Received 29 November 2013 Received in revised form 4 February 2014 Accepted 4 February 2014 Available online 12 February 2014 Keywords: Probes Phage display Epitope Mimotopes Mimtags Antibodies

a b s t r a c t In recent times the use of protein-specific probes in the field of proteomics has undergone evolutionary changes leading to the discovery of new probing techniques. Protein-specific probes serve two main purposes: epitope mapping and detection assays. One such technique is the use of phage display in the random selection of peptide mimotopes (mimtags) that can tag epitopes of proteins, replacing the use of monoclonal antibodies in detection systems. In this study, phage display technology was used to screen a random peptide library with a biologically active purified human interleukin-4 receptor (IL-4R) and interleukin-13 (IL-13) to identify mimtag candidates that interacted with these proteins. Once identified, the mimtags were commercially synthesised, biotinylated and used for in vitro immunoassays. We have used phage display to identify M13 phage clones that demonstrated specific binding to IL-4R and IL-13 cytokine. A consensus in binding sequences was observed and phage clones characterised had identical peptide sequence motifs. Only one was synthesised for use in further immunoassays, demonstrating significant binding to either IL-4R or IL-13. We have successfully shown the use of phage display to identify and characterise mimtags that specifically bind to their target epitope. Thus, this new method of probing proteins can be used in the future as a novel tool for immunoassay and detection technique, which is cheaper and more rapidly produced and therefore a better alternative to the use of monoclonal antibodies. © 2014 Elsevier B.V. All rights reserved.

1. Introduction Protein-specific probes used for epitope mapping and protein-tagging studies have been the focal point of research during recent years employing various methods of detection of proteins, such as antibodies and labelled proteins. Currently, protein analysis consists of immunoassay techniques such as ELISA, polyacrylamide gel electrophoresis and related blotting techniques that use antibodies for detection and interactions with antigens (Westermeier and Marouga, 2005). The advent

⁎ Corresponding author at: NeuroAllergy Research Laboratory (NARL), School of Life and Environmental Sciences, Faculty of Science, Engineering and Built Environment, Deakin University, 75 Pigdons Road, Waurn Ponds, Victoria 3216, Australia. Tel.: +61 3 5227 2886. E-mail address: [email protected] (C. Suphioglu).

http://dx.doi.org/10.1016/j.jim.2014.02.001 0022-1759/© 2014 Elsevier B.V. All rights reserved.

of monoclonal antibodies with the development of hybridoma technology by Kohler and Milstein in 1975 was another remarkable milestone, which has revolutionised the way we conduct research today (Borrebaeck, 2000; Chames et al., 2009; Kennett, 1979; Kohler and Milstein, 1975). Monoclonal antibodies are generally specific to the target antigen and are often used for epitope mapping of proteins for exploring the human proteome for diagnostics and therapeutic purposes (Rockberg et al., 2008). The epitope region can either be linear or conformational in their structure and the amino acid sequence can be continuous or discontinuous (Siddiqui, 2010). The ability of monoclonal antibodies to detect a single antigenic determinant (epitope) has earned it a major advantage over the use of polyclonal antisera. However, there are disadvantages of using antibodies where precise recognition of epitopes is seldom accurate (Rowley et al., 2004) and there are limitations and

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disadvantages with these techniques (Attarwala, 2010). The hybridoma technology involves many phases from the fusion of the primed B cells with myeloma cell line to the large-scale production of monoclonal antibodies from these highly selective hybrid cell lines (Falkenberg, 1998; Kohler and Milstein, 1975). For researchers working in the field, the use of this available in vitro technique is often problematic and has found to be very labour intensive and expensive (Falkenberg, 1998). Animal ethics is a major concern when it comes to development of monoclonal antibodies, which causes unnecessary suffering of animals (Falkenberg, 1998; Festing and Wilkinson, 2007). Another disadvantage of larger protein based probes, such as biotin binding proteins, is the size of these antibodies that are tagged with a detectable label. Larger probes present a challenge of inefficiently penetrating the conformational structures of target proteins as opposed to short peptides. For example, a large protein such as an antibody may interact with random functional groups on a single protein to the exclusion of thousands of other competing cytoplasmic proteins, nucleic proteins, carbohydrates and small molecules (Chen and Ting, 2005). It is therefore imperative to find new techniques that are particularly important for quantitative proteomics, less tedious and cost-effective. A more contemporary pattern of research has brought short peptide sequences under the spotlight as target specific probes for in vitro and in vivo analysis of protein ligands such as transmembrane proteins. This type of epitope tagging has led to very successful epitope mapping of target antigens. The methodology has been used for protein localization, immunoprecipitation, and protein–protein interaction (Hernan et al., 2000). Briefly, an epitope is a sequence of amino acids in any structure that is recognised by an antibody. A single protein may carry multiple epitopes that can be recognised by antibodies. A mimotope (peptides mimicking epitopes) is a structure that mimics the structure of an epitope. Due to this structural similarity, a mimotope can elicit the same antibody response as an epitope via the paratope region of the antibody. Two decades ago it was realised that using peptide phage libraries, mimotopes could be isolated which mimic the epitope on a given protein. Using this technique, the 3-dimensional structure of the epitope can be determined along with the critical residues that make up the epitope (Xu et al., 2004). Hence, in this case the mimotope acts as a probe against the desired epitope on a protein or antibody as a therapeutic agonist or antagonist. A new term introduced in this paper is ‘Mimtags’ which are the same short sequences of amino acids that play a critical role in the advancement of phage technology from a very different perspective. These short sequences of peptides are a part of fused proteins on the surfaces of phage used in phage display libraries. These mimotopes (Knittelfelder et al., 2009) that can be tagged (mimtags) on to protein sequence can effectively identify the epitope region of a target antigen as protein-specific probes. Hence, the term “mimtag” will now be used throughout the entire body of this paper replacing the term peptide mimotope. The technique has been used in various studies to prove its effectiveness. A recent study conducted by our NeuroAllergy Research Laboratory (NARL) has shown remarkable results (Ahmed et al., 2011, 2012, 2013). In our study, random peptide library on M13 phage was used to screen IL-4R and IL-13. An

affinity selection in this way allows the identification of mimtags that bind to the cytokine and its receptor, and hence, will most likely target epitopes more effectively due to the small size and nature of the mimtag (12-mer peptide). The mimtag also has the ability to conform to 3-Dimensional configuration, increasing its binding efficiency (Bredehorst and David, 2001). The bound peptide was sequenced and synthesised for further characterisation by immunoassay techniques. The study shows the potential use of biotinylated peptide mimtags as examples and their advantage over complex antigens and antibodies being applied as novel protein specific probes. 2. Materials and methods To reveal the methodology involved in the isolation and characterisation of novel mimtags, we have divided the methods between two mimtags denoted N1 as an antagonist for IL-4R and P9 for IL-13. Phage display along with ELISA immunoassay (for the N1 mimtag) and dot-blot assay (for the P9 mimtag) were used for immunological characterisation of mimtags. Our research group identified both mimtags using phage display technology and inhibited the proteins from binding to their corresponding proteins in the case of IL-4Rα (BioScientific Pty Ltd, NSW, Australia) with cytokine interleukin-4 (IL-4) and IL-13 (Abcam, Cambridge, UK) with interleukin-13 receptor (IL-13R). Two different techniques have been used with 2 different proteins to show a range of method applications with this technology. 2.1. General description of the M13 phage library kit Phage display libraries, PhD-12, in which 12-mer random mimtags are expressed at the amino terminus of protein pIII of the filamentous bacteriophage were purchased from New England BioLabs Inc. (Beverly, MA USA), as a kit. The peptide mimtag is followed by a short spacer sequence Gly-Gly-Gly-Ser, followed by the wild-type pIII sequence. Each of the libraries had a complexity of 2 × 109 virions and was amplified to give a final titer of 2 × 1011 plaque-forming units (pfu). All experiments were carried out under strict sterile conditions using a Class II biological safety cabinet. Phage display peptide library (on arrival, the kit components were stored at − 20 °C): - 100 μl, ~ 1 × 1013 pfu/ml. Supplied in TBS with 50% glycerol; - 96 gIII sequencing primer (5′-HO CCC TCA TAG TTA GCG TAA CG-3′, z100 pmol, 1 pmol/μl); - 28 gIII sequencing primer (5′-HO GTA TGG GAT TTT GCT AAA CAA C-3′, 100 pmol, 1 pmol/μl); - Escherichia coli (E. coli) ER2738 host strain; F′ proA + B + lacq-(lacZ) M15 zzf::Tn10 (TetR)/fhuA2 glnV thi(lac-proAB)-(hsdMS-mcrB)5 (rk mk McrBC). Host strain supplied as 50% glycerol culture: not competent. Stored at − 20 °C. 2.2. Immobilization of target IL-4Rα and biopanning The target solution was prepared using 0.1 M sodium hydrogen carbonate (NaHCO3, pH 8.6), mixed with 5 mg/ml

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bovine serum albumin (BSA) and 0.02% sodium azide (NaN3). This solution is called the blocking buffer, and was filter sterilized before diluting the target. Two separate concentrations of the target IL-4Rα were produced initially, 50 nM and 10 nM. Once the concentrations were prepared, they were aliquot in small volumes into Eppendorf tubes and stored at − 20 °C until needed. Five consecutive rounds of biopanning were performed. A few minor stringency changes were made at each round to select phage with maximum binding to the target. The following panning procedure pertains to round 1. 150 μl of the target protein IL-4Rα was added to wells on the microtiter plate. The coated plate was covered with parafilm and incubated overnight at 4 °C. Following day, the solution from the wells was discarded. The wells were filled with blocking solution (BSA + NaN3). The plate was then incubated for an hour at 4 °C. Post-incubation with the blocking solution, the plate was washed 6 times with 200 μl TBST (TBS; 50 mM Tris–HCl pH 7.5, 150 mM NaCl + 0.1% [v/v] Tween-20). Between each consecutive washing, the TBST was discarded. 10 μl of M13 from the Ph.D. 12-mer phage display library (containing 4 × 1010 pfu and ~55 copies of each 12-mer peptide sequence) was diluted with 90 μl of 0.1% TBST. 100 μl of this dilution was pipette into a coated plate well and rocked gently for an hour on a platform rocker, at room temperature. Following incubation of the phage, the library solution was discarded. Six TBST washes were rapidly performed to wash off unbound phage or phage that was bound non-specifically to the target antigen. To elute the phage that specifically bound to the target receptor, a buffer for nonspecific disruption of binding was used. The buffer was 0.2 M glycine HCl (pH 2.2) along with 1 mg/ml of BSA. 100 μl of this buffer was added to the wells of the microtiter plate and was rocked gently for no more than 10 min. The eluate was pipetted into a microcentrifuge tube, and neutralised with 150 μl of 1 M Tris–HCl, pH 9.1. At this stage, the phage eluate is not amplified, but simply obtained from panning. The unamplified phage stock, roughly 250 μl, was stored in the fridge at 4 °C (in the dark) to be used later. The TBST Tween-20 concentration (v/v) for consecutive rounds was increased gradually to perform more stringent washes and select for strong interactions between the peptide and the target receptor. In addition, the concentration of target IL-4Rα was reduced in subsequent rounds. 2.3. Amplification and identification of eluted phage clones The titer of the unamplified phage stock was mixed with a freshly prepared culture of Escherichia coli (E. coli) ER2738 (Phage Display Kit; New England Biolabs) host strain in 10 ml of LB medium, which is tetracycline resistant (TetR), and incubated in a rotator shaker at 37 °C, while shaking at 240 rpm, for 4.5 h. The culture was then transferred to a centrifuge tube and spun for 10 min at 10,000 ×g at 4 °C, to pellet bacterial cells. The supernatant (containing the amplified phage) was transferred to a fresh tube and re-spun for 5 min. Pellet was then discarded. Next, 80% of the upper supernatant was transferred to another fresh centrifuge tube. The phage was precipitated by using 20% (w/v) PEG/2.5 M NaCl, or roughly 1/6th of the total volume of eluate recovered from amplification. Dilutions of the phage were prepared and grown on plates containing luria broth

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(LB) along with isopropyl β-D-1-thiogalactopyranoside (IPTG) and 5-bromo-4-chloro-3-indolyl-β-D-galactopyranosideplates (X-gal) (LB medium + 15 g/l agar + 1.25 g of IPTG + 1 g X-gal in 25 ml of dimethyl formamide; Sigma Aldrich). The library-cloning vector M13 is derived from the common cloning vector M13mp19, which carries the lacZα gene. This results in phage colony plaques appearing blue when plated on media containing X-gal and IPTG (Kopp et al., 2007). Phage was sequenced from randomly selected clones as described in the phage manual provided by New England Biolabs (Devlin et al., 1990; Gazarian et al., 2000; Gnanasekar et al., 2004; Smith et al., 2005; Tikunova and Morozova, 2009) from the Micromon DNA Sequencing Facility at Monash University (Melbourne, Australia). The mimtag sequences were deduced from the obtained oligonucleotide data and aligned for homologies by means of Finch TV version 1.4.0 software indicating the 5′ prime and 3′ prime ends (Lau and Robinson, 2009). A total of 10 mimtags were determined from the total pool of available motifs. This helped ascertain which sequences out of the 10 were most suitable to carry out further immunoassays and had 100% homology in their nucleotide sequences of their corresponding proteins N1 with IL-4 and P9 with IL-13R. The flanking regions were used to locate the 36 base pairs of DNA of interest, which encoded the 12-mer peptide sequence displayed on the M13 phage surface. The hybridization positions of the -28 and -96 sequencing primers were indicated as well. Short mimtags designated N1 and P9, along with their biotinylated versions, were synthesised by AUSPEP (Melbourne, Australia) and received in a lyophilised condition. The biotin was added to the N terminus of the mimtags. The quality of all the mimtags was assessed by high performance liquid chromatography (HPLC) and confirmed by mass spectrometry analysis (purity N 90%). Further analysisincluded alignment of 12-mer sequences with IL-4 cytokine and IL-13R using ClustalW2 multiple alignment software. 2.4. M13 binding ELISA Four rows of a 96-well microtiter plate were coated with 90 μl target protein, IL-4Rα (10 nM). The plate was incubated at 4 °C (in a humidified box) with gentle agitation. Following day, the excess target solution was discarded. The wells containing the target proteins were blocked using blocking solution (5 mg/ml bovine serum albumin (BSA) and 0.02% sodium azide (NaN3)). Simultaneously, a second microtiter plate, without the target proteins, was also blocked with the blocking solution. Both plates were sealed and incubated at 4 °C for 1–2 h. Once again, the excess blocking solution was discarded. Each plate was washed 5 times using 0.3% TBST. 4-fold serial dilutions of the phage clones (N4, N5 and N7) in 200 μl of 0.3% TBST were prepared; starting with 1012 pfu in the first well, down to 2 × 105 pfu in the 12th well. Phage dilutions from 4th, 5th, 6th, 7th and 8th wells were transferred to the plate with the target proteins. This was incubated for 1–2 h at room temperature (RT), with agitation. Once again, the plate was washed 5 times with TBST. 200 μl of primary antibody (mouse anti-M13 antibody (Sigma Aldrich), 1:1000 dilution in blocking buffer) was added to each well and incubated for 1 h at RT. Subsequently, the plate was washed 5 times with TBST.

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Post-incubation with anti-M13 antibody, 200 μl of secondary antibody (anti-mouse horseradish peroxidise (HRP) conjugated antibody (Sigma Aldrich), diluted 1:2000 in blocking buffer) was added to each well and incubated for 1 h with agitation on a rocker. This was followed by another round of 5 washes with 0.3% TBST. A developing solution was prepared by dissolving a single tablet of phospho-citrate buffer with sodium perborate in 100 ml of deionised water (dH2O), and then 1 tablet of HRP substrate ortho-phenyl diamine (OPD; Sigma Aldrich) was dissolved in 7.5 ml of this solution. 200 μl of this solution was added to each well; the plates were wrapped in foil and incubated for 30 min at room temperature with agitation. Adding 50 μl of 4 M H2SO4 stopped the reaction. The colour development on the plates was read by using Thermo Labsystems Multiscan plate reader, set at 492 nm of wavelength. 2.5. Application of mimtag technology using direct ELISA Immobilization of 90 μl target IL-4R (10 nM) was carried out on a 96 well plate overnight. The plate was sealed with a parafilm and placed in a humidified box with agitation. Following day, the target solution was discarded. 220 μl of blocking buffer (BSA + NaN3 blocking solution) was added to the wells. The plate was incubated at 4 °C for 1–2 h. The blocking buffer was then discarded and the plate was gently washed 3–4 times with 280 μl/well of 0.3% TBST. Next, 100 μl of biotinylated N1 mimtag was added to each designated well of the ELISA plate. The plate was incubated for 1–2 h at RT with agitation on a rocker. Negative controls were set up in 3 separate wells without the addition of N1 mimtag. Finally, 150 μl HRP conjugated streptavidin (Sigma Aldrich) (1:500 dilution in blocking buffer) was added to the wells and incubated for 1 h with agitation on a rocker. Once again, the plate was gently washed 3–4 times with 280 μl/well of 0.3% TBST. A developing solution was prepared as described in Section 2.4. 200 μl of this solution was added to each well; the plates were wrapped in foil and incubated for 30 min at RT, with agitation on a rocker. Adding 50 μl of 4 M H2SO4 stopped the reaction. The colour development was detected using a plate reader at 492 nm. 2.6. Application of mimtag technology in a dot-blot assay The target protein (recombinant human IL-13) was immobilized on two 1 cm × 1 cm nitrocellulose membranes (Sigma Aldrich) by adding 5 μl of IL-13 at 50 nM concentration. The membranes were air dried for approximately 10 min before being blocked by 500 μl of blocking buffer. The membranes were washed three times using 0.15% TBST in a weighing boat with shaking on an orbital shaker. One membrane was then incubated with 1 ml of 0.1 mg/ml biotinylated mimtag P9 for 1.5 h at RT on a rotary wheel. The other membrane was incubated with sterile dH2O as a negative control. The membranes were washed again in separate weighing boats and then incubated with 1 ml of HRP-conjugated streptavidin (1:500 diluted in PBS) for 1 h at RT on a rotary wheel. The membranes were washed four times with 0.15% TBST. The membranes were then incubated with the chemiluminescent HRP substrate (HRP substrate peroxide solution/HRP substrate luminal

reagent) for 30 s and the membrane images were immediately captured on a Fujifilm Chemidoc System. 3. Results Peptide phage clones from 12-mer libraries were analysed by taking 3 random peptides from the 10 individual phage clones isolated from the 5th round of biopanning. Each clone was diluted, plated, amplified, sequenced and tested for reactivity with the target protein IL-4R using ELISA immunoassays and in the case of IL-13, a dot-blot (Fig. 1). A different technique to the different proteins has been used to show a range of method applications. 3.1. Phage display; biopanning A 12-mer phage-displayed random peptide library was screened through a process known as ‘biopanning’ as described in Sections 2.1 and 2.2 above. The procedure was performed with five cycles, repeated consecutively. Initially, the first round of biopanning was completed and the amplified phage was titered onto IPTG/X-gal Plates. A total of 5 rounds of biopanning were repeated. Table 1 shows the calculations done to obtain input volumes for subsequent panning rounds. From the final round of biopanning, 10 clones were collected by stabbing plaques on the agar plates and amplified as described in the Phage Display Manual (New England Biolabs). The phage clones were sequenced to reveal the genome of the single-stranded phage DNA. Upon DNA sequencing, 9 of the 10 sequences had identical consensus in binding sequence for IL-4R (N1 to N9). Since the mimtags from the clones N1 to N9 showed a consensus in sequence, N10 was discarded and was no longer used for further analysis. In a similar fashion, the P9 mimtag (for IL-13) was selected out of 10 mimtags with identical sequences, which showed similar amino acid sequence with IL-13R. Further, ClustalW2 was used to perform sequence analysis of N1 amino acid with the IL-4 cytokine to observe the similarities between their sequences. This was performed to evaluate the binding capacity of the mimtag to IL-4R, and as a result, “mimic” the cytokine IL-4 in its amino acid sequence. This also revealed the epitope region of specific amino acid sequence of IL-4R that would interact with the mimtag. Table 2 shows a comparison between IL-4 and the N1 mimtag with 6 amino acid residues showing identical residues, and also three weakly conserved residues. 3.2. M13 ELISA with three homologous M13 phage clones M13 ELISA was performed using 3 selected phage clones N4, N5 and N7 from the 9 clones with identical sequences selected for mimtag synthesis. This step was performed to further enhance results showing specific binding of the phage mimtag to the target IL-4R. Irrespective of which phage clones were used for the M13 ELISA, since all of them displayed the same amino acid sequence, the ELISA was carried out using an anti-M13 antibody, followed by an anti-mouse HRP antibody to detect antibody-binding and OPD substrate for HRP, to develop the solution for absorbance detection (Fig. 2). The detection was based on a strong affinity between the displayed mimtag by the phage clones

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Fig. 1. Schematic diagram of the overall steps taken to ensure the isolation and testing of the mimtags. (a) Phage display was performed on the target proteins (IL-4R or IL-13) to isolate the mimtags after 5 rounds of biopanning. (b) Phage grown on LB agar plate by infecting E. coli and 10 individual blue phage clones isolated, amplified and purified for use in DNA sequencing (c) and M13 ELISA (d). Specific assays to confirm binding of the mimtag to its target proteins include a direct ELISA (e) or a dot-blot (f), both utilising a biotinylated version of the mimtag.

and the target IL-4R. Overall, all of the three selected peptides (e.g. N4, N5 and N7) showed a binding to the IL-4R target. With comparable error bars overlapping between the 3 different clones, shown in the graph (Fig. 2), the phage clones displaying the mimtag gave higher absorbance readings when compared to the control. This is an indication of the accuracy and specificity of the M13 phage binding to its target antigen in vitro. 3.3. Direct ELISA In view of the fact that N1 amino acid sequence shared a 50% identity with cytokine IL-4, it was necessary to conduct Table 1 Input titers for subsequent rounds of biopanning and calculations. Biopanning rounds

Initial pfu

Input titer of phage

Input volume for next round

Biopanning round 1 Biopanning round 2 Biopanning round 3 Biopanning round 4 Biopanning round 5

2.25 × 1011

2 × 1011/2.25 × 1011

0.88 μl

2.3 × 1010

2 × 1011/2.3 × 1010

8.8 μl

5.8 × 1011

2 × 1011/5.8 × 1011

0.344 μl

1.3 × 1011

2 × 1011/1.3 × 1011

1.3 μl

Last round, hence no further calculations required

further tests on the N1 biotinylated mimtag, to determine its interaction with IL-4R. This was performed by using direct ELISA, in which the mimtag was allowed to interact with the IL-4R in the absence of the cytokines to determine its binding to the target antigen. In a similar fashion as other ELISA assays, this was carried out using a HRP-conjugated streptavidin, to detect the biotin on the N1 mimtag, and OPD substrate to develop the solution for absorbance detection as described in earlier sections. Evidently, the differences in absorbance readings obtained showed that N1 mimtag had specifically bound IL-4R, when compared to the negative control. A trend can be seen in the graph (Fig. 3) with increasing concentration of the mimtag, higher absorbance readings were recorded on the spectrophotometer. This indicated that the streptavidin-HRP had indeed recognised the biotin on mimtag N1, which has interacted with the IL-4R target protein. Table 2 Results obtained from ClustalW2 alignments, performed using the N1 mimtag. Percentage similarity with 12 amino acid sequence of N1 peptide

With IL-4

With peptide

Identical sequences Strongly conserved Weakly conserved

6 amino acids (50%) None 3 amino acids (25%)

100% 100% 100%

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Fig. 2. M13 ELISA performed using 3 selected phage clones of IL-4R. The phage clones were randomly selected from the 9 clones with identical sequences to verify the binding affinity and efficacy of the mimtag to the target IL-4R protein. Figure illustrates the absorbance readings that were obtained when a range of phage dilutions were used to perform the experiment. As a result, with increasing phage clone titers, a higher absorbance reading was obtained at 492 nm, and vice-versa. The results represent 3 replicates (mean values) for each bar graph; hence the error bars denote a standard deviation. A statistical Student's t-test revealed a significant difference (*) between the mean absorbance values of the 1.5 × 1010 phage dilution and the negative control (P b 0.05).

3.4. Dot-blot analysis of mimtag binding to target antigen (IL-13) In order to test a different application method with a different protein, nitrocellulose membrane was spotted with IL-13 and incubated with the mimtag peptide, showing a dark spot that indicates binding between the immobilized IL-13 and the mimtag P9 (Fig. 4). The nitrocellulose membrane without the peptide gave a plain white image when analysed by the chemidoc equipment. The “test” nitrocellulose membrane, in which only one side was immobilized with the target protein

Fig. 3. Direct ELISA of biotinylated N1 mimtag interaction with IL-4R. This assay was conducted to verify the affinity and efficacy of the newly synthesised N1 mimtag, which showed considerable binding with the IL-4R target. The graph illustrates the absorption readings that were obtained when a range of N1 mimtag concentrations were used to carry out the experiment in vitro. As a result, with increasing mimtag concentrations, a higher absorbance reading was obtained at 492 nm, and vice-versa. The results represent 3 replicates (mean values) for each bar graph; hence the error bars denote a standard deviation. A statistical Student's t-test revealed a significant difference (*) between the mean absorbance values of the mimtag at 0.2 mg/ml concentration and the negative control (P b 0.05).

Fig. 4. Dot-blot analysis of the binding affinity of the biotinylated mimtag P9 to IL-13. Nitrocellulose-membrane-immobilized IL-13 (target protein) was incubated with and without the mimtag to show interaction between the two molecules. Binding is depicted by the dark circle in the middle of the nitrocellulose membrane treated with the mimtag P9.

IL-3, showed a dark dot on the side with the target protein, whereas the other side remained plain with no colouration. 4. Discussion The aims of this work were, to isolate phage-displayed mimtags against IL-4R and IL-13, to investigate the ability of mimtags to bind target proteins and act as protein-specific probes. High-quality mimtags were isolated during the study, which recognised IL-4R and IL-13 specifically. 4.1. The use of phage display in isolating mimtags as protein specific probes Once again, for discussion purposes only, mimtag N1 will be emphasised throughout this section. New strategies have been devised in the last few years to characterise regions with epitope binding capacity. Phage display has shown to be a powerful tool to discover and unravel conformational mimic epitopes or mimotopes from random peptide libraries (Luzzago et al., 1993; Pacios et al., 2008). In this study, phage display was successfully used to identify mimtags for target proteins IL-4R and Il-13 cytokine. 12-mers are long enough to fold into short structural elements, which may be useful when panning against targets that require structured ligands. The exposed random peptides were allowed to interact with the sequences of the IL-4R. The purity and efficacy of the target was determined by the company (Sigma-Aldrich), which certified the human IL-4R to be pure and biologically active. A 12-mer library was used in the assay as it renders peptides that include highly reactive mimotopes as opposed to 7-mer libraries (Gazarian et al., 2000). The additional 4th and 5th round of selective biopanning discarded almost completely the low affinity binders and 9 out of the 10 mimtags isolated had a consensus in their amino acid residues. In a similar fashion, the most suitable candidate P9 mimtag was isolated against IL-13 cytokine using 5 rounds of biopanning. The use of phage display has many advantages over the conventional system of using antibodies for the detection of epitopes. Many studies over the years have shown the usefulness of phage display in the field of research. The possibility of obtaining mimtags of interest without learning

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the three-dimensional structures of proteins avoids the need for complicated cloning, crystallizing, and modelling procedures, which had to be carried out first (Furmonaviciene et al., 1999; Ganglberger et al., 2000; Jensen-Jarolim et al., 1999; Riemer et al., 2004). The in vitro analysis using a 12-mer phage display peptide library eliminates the use of animals for the production of antibody libraries, which subsequently eliminates any issues relating to animal ethics and keeps animals out of harm's way. Mimtags are also smaller in size when compared to antibodies, which increases their effectiveness and efficiency to penetrate large protein fragments or antigens with conformational epitopes (Knittelfelder et al., 2009). 4.2. Probing of a conformational epitope on target protein with mimtag The mimtag sequence N1 had shown to share a homology with the cytokine IL-4 at specific amino acid sequences. Before stepping into further analysis of the synthesised mimtag, it was important to understand the interaction between IL-4 and its receptor IL-4R relative to the binding of mimtag N1 to IL-4R. The multiple sequence alignment in ClustalW2 showed a 50% identity of amino acid sequences of N1 mimtag with amino acid sequence of IL-4 cytokine. There were gaps in the sequence alignments, which indicated a loop region with no secondary structure conserved. Based on hydrogen bonding, there are three clusters that form ligand receptor interface. In the IL-4R, two out of three clusters have been identified as the main binding determinants (Fig. 5), Glu9 (IL-4) and Arg88 (IL-4). The Glu9 makes several hydrogen bonds with IL-4R at positions Tyr13, Ser70 and Tyr183. The second cluster involves Arg88 of IL-4, which bonds with Asp72 of IL-4R (Kraich et al., 2006). The critical binding residues are distant in the primary sequence but close in the folded native conformation of the protein hence forming a discontinuous chain of amino acid residues (Luzzago et al., 1993; Smith and Petrenko, 1997). However, the N1

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mimtag showed binding to IL-4R in the binding ELISA assay which did not overlap the binding sequence of IL-4 cytokine and its receptor when cross-checked with ClustalW2 alignment software. Hence, it may be possible that the N1 mimtag may have identified a novel epitope of IL-4R, which remains unreported (Minton, 2008; Moy et al., 2001). 4.3. Mimtag technology Mimotope tagging is a very versatile procedure to identify and tag epitope regions of proteins using biotinylated versions of the mimotopes. Mimotopes isolated from phage display libraries enable alignments with the target antigen and subsequent localisation of structural epitope regions (Hantusch et al., 2004). Another useful outcome of such a study is the use of such mimotopes to tag target proteins, which allows them to be used as protein-specific probes, as in the case of mimtags N1 and P9. There are several advantages of this system over the conventional use of antibodies as protein specific probes. Mimtags can be stored over long periods of time without the fear of deteriorating and are cheaper to produce compared to monoclonal antibodies. They are relatively free of contaminants such as toxic substances from expression systems. Application based advantages include the high-throughput of peptide mimtags, which allows the simultaneous bombardment of the target antigen with several billion mimtags and this allows the interaction of the target protein with the best possible sequence of mimtag. No re-probing is required, as in the case of antibodies because all immunoassays can be performed with the new mimtag as demonstrated with the M13 phage ELISA, direct ELISA and dot-blot analyses. Antibodies are difficult to produce employing cell-based and in vivo methods, not to mention, the purification of antibodies (Ma et al., 2010; Tan et al., 2012), which are highly expensive methods and difficult to handle leaving very little margin for errors. The specificity is another factor that most scientists fail to

Fig. 5. Cluster domains of the IL-4R indicated by the dotted Y shape. These clusters are responsible for binding with the IL-4 cytokine. Two clusters denoted Cluster I and Cluster II have shown to bind to IL-4 cytokine with about 80% of the total binding energy. At amino acid positions 13, 70 and 183 lays the first binding region with amino acid at position 9 (glutamate residue). At amino acid position D72 lays an aspartic acid that binds with arginine at position number 88 of the IL-4 cytokine. Adapted from Kraich et al. (2006).

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report when using antibodies, which has to be determined first, making it ever more difficult to use in immunoassays and detection systems (Kobayashi et al., 1988; Saper, 2009). However, mimtags can conform 3-dimensional structures and can specifically bind and localise an epitope. Hence, we can propose that mimtags can eventually replace antibodies for their use in immunoassays and detection systems. Another important feature of using biotinylated mimtags is the spacer between the mimtag and biotin, which reduces steric hindrance to binding with the target epitope as opposed to a labelled antibody where conjugation reaction has shown to cause deleterious effects on antibody avidity (Vira et al., 2010) and in some cases, labelling index in antibody was negatively correlated with the binding affinity for its target antigen (Takai et al., 2011). A HRP-conjugated streptavidin protein can detect biotin on the mimtag with high affinity and avidity. This immunoassay was specifically done to test the efficacy and affinity of the newly synthesised biotinylated mimtag N1 to the IL-4R (direct ELISA) and P9 to IL-13 (dot-blot). The trend is clearly visible via the graphed readings where higher absorption readings are observed with increasing peptide concentrations. A Student's t-test analysis of the results from the graph revealed a significant result when compared to the control (P b 0.05). This was an indication that the IL-4R was bound to the bottom of the well and the N1 mimtag was interacting with the target. In the case of the P9 mimtag, this was further supported by the result of the dot-blot analysis, which showed binding between the mimtag and IL-13. Thus, a range of detection application methods can be used with mimtags. 4.4. Further applications of mimtag technology Apart from their ability to probe and map protein sequences, mimtags can potentially be used for probing and identifying gene sequences and clusters; using a phage library of exon-sized inserts (Fehrsen and du Plessis, 1999; Mullaney et al., 2002; Tungtrakanpoung et al., 2006). Mimtags can also be used for the detection of antibodies in a given sera which is by far an inexpensive, rapid, sensitive, specific and reusable method to detect a humanised therapeutic antibody (Shang et al., 2011). Phage displayed synthetic mimtags have also been used for biosensor analysis by fluorescently labelling the mimtags that could be detected onto a surface of a sensor chip designed for ligand interaction and replacing larger antibodies as probes (Goldman et al., 2000; Pavan and Berti, 2012). Single point substitution in mimtag sequences can be performed to improve the avidity of the mimtag enhancing its bioactivity and interacting capability with the target epitope (Magliani et al., 2004; Saphire et al., 2007). All of these applications are just a start into replacing larger antibodies with smaller mimtag protein-specific probes for the detection and labelling of key epitope regions on a given protein with higher specificity and low costs making it a more sophisticated and effective method. 5. Conclusion We have successfully demonstrated that both examples of IL-4R and IL-13 as target proteins illustrate the importance of using mimtags in the field of proteomics as protein-specific probes along with many other examples (Knittelfelder et al.,

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