Screening of peptide receptor sequences for amitrol detection using chromatographic biopanning - Nguyen Thi Lien Thuong

Journal of Science and Technology 54 (2A) (2016) 6-13 SCREENING OF PEPTIDE RECEPTOR SEQUENCES FOR AMITROL DETECTION USING CHROMATOGRAPHIC BIOPANNING Nguyen Thi Lien Thuong 1, * , Nguyen Thanh Binh 1 , Yoo Ik-Keun 2 1 Faculty of Resources and Environment, Thu Dau Mot University, No 6 Tran Van On street, Phu Hoa ward, Thu Dau Mot city, Binh Duong province, Vietnam 2 School of Chemical engineering and Bioengineering, University of Ulsan, 3 Daehak-ro, Nam-gu, Ulsan, South Korea * Email: lienthuong@gmail.com Received: 1 April 2016; Accepted for publication: 15 June 2016 ABSTRACT Endocrine disrupting chemicals (EDCs) have been known as health threatening compounds and much attention has been paid in a research related with detection and/or removal from food sources and living environment. Amitrol, classified as one of EDCs, is hard to be detected and removed due to its chemical characteristics such as small molecular weight, low reactivity and high solubility. Some of organic compounds were investigated to find the corresponding binding peptides to those by biopanning protocol. However, peptides with affinity to amitrol remained to be searched for because amitrol is hard to be used as a target of free form or immobilized form in common biopanning protocol. In our experiment, amitrol was successfully immobilized on CDI monolithic column with the presence of catalyst and hence was able to be applied as a target component in biopanning. Chromatic biopanning was carried out as a fast and convenient method for the selection of peptides with high affinity to amitrol. Chemical and physical methods were combined in elution step to improve the selection efficiency for strong binders. After multiple rounds of negative screening and positive screening, high affinity peptide sequences were isolated from initial peptide libraries. Applications of these peptide sequences can be widened from bio adsorption to biosensor for environment pollution control. Key words: Amitrol, phage display, chromatographic biopanning 1. INTRODUCTION Amitrol is a nonselective systemic triazole herbicide used on non-cropland for control of annual grasses and perennial and annual broadleaf weeds, poison ivy and aquatic weeds in marshes and drainage ditches [1]. All use of amitrol on food crops was canceled by the EPA in 1971 for having caused cancer in experimental animals [2]. Amitrol is a compound of very low acute toxicity to humans and other animals. Associated symptoms in humans include skin rash, vomiting, diarrhea, and nose bleeds. Poisoning of several species by amitrol is characterized by Screening of peptide receptor sequences for amitrol detection 7 increased intestinal peristalsis (this may lead to diarrhea), fluid in the lungs, and hemorrhages of various organs. A single dose of 1,200 mg/kg amitrol reduced iodine uptake by the thyroid in healthy persons [3- 5]. The maximum residue levels of pesticides in water should not exceed 0.1 μg /l according to the European Union. However, analysis of amitrol in water at low concentration is very difficult due to it high solubility in water, low solubility in non-water miscible organic solvents. Currently there is no efficient method for extracting it from aqueous samples to increase its concentration to a detectable level. Analysis of amitrol is also a problem. Due to its high polarity and low volatility, small extinction coefficient, currently method such as gas chromatography and liquid chromatography face difficulties either. The most effective method for analyzing amitrol now is using capillary electrophoresis with detection limit of 4 μg /l. This method also has to couple with evaporation of water sample before analyzed by CE [1, 6]. So it’s important to have a sufficient method for the detection of amitrol. Currently there is not yet an effective way for removal amitrol. Specific peptide sequences from biopanning are known to have high affinity toward organic targets and applied as sensor receptors or bio-absorbents. Considering this characteristics of peptide, the usage of organic- binding peptide would be useful strategy for the removal of amitrol at low concentration from aquatic environment. Phage display of peptide libraries is a selection technique which enables the screening of phage possessing specific affinity to a given target molecule due to peptides expressed as a part of surface proteins. This screening process is called biopanning and has become the standard technique for obtaining peptides cognitive of specific molecules. For example, phage display was used to find heptapeptides with affinity to cadmium ion, lead, bisphenol A as well as develop into biosorbent for pollutant removal [7, 8]. These target chelating peptides isolated could be inserted onto bacteria surface and showed high tolerance to toxic compound. In this study, we applied chromatographic biopanning, which has been successfully used to screen for high affinity peptide sequences to toxic target, to screening for peptide receptor sequencing with affinity to amitrol. The combination of chromatographic biopanning, strong washing buffer strength and sonication elution was studied to achieved the expected good sequences and shortens the screening process. 2. MATERIALS AND METHODS 2.1. Immobilization of amitrol in CIM ® CDI FPLC column Amitrol (Sigma, Aldrich) was immobilized to CDI groups on CIM-CDI column (approximately 0.6 mmol /g support according to the product information provided by BIA Company), and imidazole.HCl was used as catalyst. The amount of amitrol reacted was calculated by subtracting the amount of input with remain amitrol after the reaction and was analyzed by HPLC. Amitrol analysis: HPLC column: amine bonded Luna 5 m NH2 100A. Flow rate 1.5 ml min. Detection at wavelength of 220 nm. Mobile phase Isooctane/ethanol (60:40) was used. 2.2. Phage tittering and amplification 2.2.1. Phage display peptide library Nguyen Thi Lien Thuong, et al. 8 A phage display peptide library with disulfide bonded constrained hepta-peptide from NEB (E8120S, New England Biolabs, Beverly, MA) was used to search for phage with high affinity to amitrol (Figure 1). The library complexity is 1.2 x 10 9 sequences [10]. Figure 1. Phage M13 with displayed constrained peptide and spacer. 2.2.2. Phage tittering and amplification Mediums and chemicals for phage titering and phage amplification: LB, LB-tet, LB agar plate 2YT, IPTG /Xgal, top agarose, PEG /NaCl. Method for tittering and amplification of phage was mentioned in the protocol from NEB [9]. 2.3. Chromatagraphic biopanning using Fast Protein Liquid Chromatography The chromatography system used was AKTA primer plus (GE Healthcare). The data was processed with PrimerViewer software. Bare CIM®CDI column (214.8000, BIA Separations) and CIM®CDI column immobilized with amitrol were used for chromatographic biopanning [10]. Chromatographic biopanning procedure was shown in Figure 2. FPLC buffer: Buffer used in the experiment included equilibration buffer (phosphate saline buffer 20 mM, NaCl 0.5 M, pH 8.0), washing buffer 1 (phosphate saline buffer 20 mM, NaCl 0.5 M, pH 8.0), washing buffer 2 (Glycin.HCl 0.08 M, pH 3.6), washing buffer 3 (Glycin.HCl 0.2 M, pH 2.2) and elution buffer (Glycin.HCl 0.2 M, pH 2.2) 2.3.1. Negative screening with bare CIM®CDI column CIM®CDI disk (CV=0.34 ml) was used in negative screening round. Equilibration buffer (phosphate saline buffer 20 mM, NaCl 0.5 M, pH 8.0) was used to equilibrate the column. A 100 μl of phage solution (1012 phages, complexity about 109) was load to the column through inlet line with flow rate at 1 ml /min for 25 min. 2.3.2. Main biopanning with CIM-CDI-Amitrol column One CIM®CDI -amitrol disk was used to select phages with affinity to amitrol. A 200 μl of phages (~10 12 virions) amplified from phage of negative screening. After sample loading, 10 ml of washing buffer 1 (pH 8.0) was used to wash away phages bound weakly to the column at flow rate 2 ml /min. Second washing step was done with 40 ml of washing buffer 2 (pH 3.6) at flow rate 2 ml /min to remove stronger bound phages. Third washing step was followed continuously with 40 ml of washing buffer 3 (pH 2.2) at flow rate 1 ml/min. The CIM®CDI-Amitrol disk was then placed in a sterile tube containing 4 ml of elution buffer and sonicated for 2 × 30 seconds to collect phage. Phages from elution step were amplified for next round of biopanning. Second round of biopanning was carried out in the same protocol with first round. Screening of peptide receptor sequences for amitrol detection 9 Figure 2. Overview of chromatographic biopanning process with negative screening against bare monolithic column and main biopanning against amitrol immobilized on monolithic column. 2.3.3. DNA extraction and sequencing 60 individual phage clones were isolated randomly and amplified. The phage ssDNA was purified and collected using QIA M13 kit (27704, Qiagen) and sent for DNA sequencing. A - 96gIII sequencing primer (S1259S, NEB) 5´- HO CCC TCA TAG TTA GCG TAA CG –3´ was used for reading the inserted sequence. 3. RESULTS AND DISCUSSION 3.1. Negative screening with bare CIM®CDI column To eliminate unwanted phage attached to screening materials, negative screening was carried out. Phages were added to the FPLC screening system and flow through was collected and recycled through the column for 40 minutes (Figure 3). Unbound phages having no affinity to bare column in flow through were counted and amplified to be used in next round of biopanning using amitrol target. Tittering result showed that most of library phage did not bind to column matrix. It was indicated that unbound phage having no affinity to the screening system remained around 10 12 phages on flow through and this number was almost equal to input phage amount (Table 1). Nguyen Thi Lien Thuong, et al. 10 Figure 3. Chromatographic FPLC biopanning system. 3.2. Main biopanning with amitrol immobilized on column Round 1 In the first round of main biopanning, one CIM®CDI disk with amitrol immobilized on it was used to select phages with affinity to amitrol. Results from tittering (Table 1) indicated that most of phages were removed off the column during washing step (~10 12 ) due to high strength of washing buffer. Washing step 1 (pH 8.0) and washing step 2 (pH 3.6) were applied in order to remove phage bound weakly to amitrol. Third washing step with very low pH (pH 2.2) was used to increase washing strength and remove most of bound phages, left very strong binders on the column. 10 6 phages were collected by elution with sonication and these phages were thought to have very high affinity to amitrol. Elution by sonication in elution buffer (pH 2.2) is a combination between chemical and physical method was used to collected strong binder on the column. According to the results of other groups sonication will help to select phages with high affinity to target and time effective in reducing the number of round of biopanning [6, 7]. The biopanning was also seen in figure 4, peak of input phages was seen and a broad peak in washing step indicated the presence of weak binding phage removed during this stage, while no peak was shown in washing stage. Phage was eluted gradually during elution step proven by broad peak in the chromatogram. Round 2 Phages from elution fraction of round 1 were amplified and used in second round of biopanning. In this round, the same protocol to round 1 was applied and phages from washed fraction 1 st , 2 nd , 3 rd and eluted fraction was counted by tittering. In figure 5, blue line peak of input phages was seen clearly while no peak was shown in washing stage. Phage was eluted gradually during elution step proven by broad peak in the chromatogram. The tittering results (Table 1) showed that most of input phages (~10 12 pfu) bound to the column, almost no phages was washed during three washing steps at pH 8.0, 3.6 and 2.2. Only about 10 7 phages were Screening of peptide receptor sequences for amitrol detection 11 eluted by sonication step. Phages collected from round 1 showed very high affinity to amitrol; they still remained with low pH washing buffer of round 2 and only collected by sonication. A 10 12 phage particles was applied to 1.14 x 10 20 molecules of amitrol as calculated which is much higher than phage number so all phage could bound to the column. Due to the strong affinity of phages and the structure of column, short time of sonication of 2×30 seconds was not sufficient to remove all bound phage and only ~10 7 phages were eluted by sonication. Longer sonication time in elution step may destroy the phages [7]. No weakly bound phage was washed away during washing step so main biopanning was stop at second round. This trend of screening results was also indicated in the study of Donatan et al. when applying sonication for elution step [6]. Instead of taking phage from elution fraction of round 2 in which washing steps did not help to remove any weakly bound phage, phages from eluted fraction of round 1 were randomly selected for sequencing. 2010409no001:1_UV 2010409no001:1_Cond 2010409no001:1_Conc 0.0 2.0 4.0 6.0 8.0 mAu 0.0 10.0 20.0 30.0 40.0 50.0 60.0 70.0 80.0 ml Figure 4. Main bio panning round 1 against CIM-CDI-Amitrol column. Green line represented concentration of buffer. Blue line represented UV absorbance at 280nm for phage detection and red line represented conductivity of biopanning solution. Table 1. Results from chromatographic biopanning Round of biopanning Phage applied (pfu) Flow through/ Washing (pfu) Elution (pfu) Negative screening ~1012 ~1012 Main biopanning round 1 ~1012 0.9×1012 1.6×106 Main biopanning round 2 ~1012 0 2×107 Nguyen Thi Lien Thuong, et al. 12 Figure 5. Main biopaning round 2 against CIM-CDI-amitrol column. Green line represented concentration of buffer. Blue line represented UV absorbance at 280nm for phage detection and red line represented conductivity of biopanning solution. 3.3 DNA extraction and sequencing Sequences of 12 amino acid constraint peptide having strong affinity to amitrol were obtained as shown in Table 2. The three sequences CSATPRPSC, CLIPTQAMC, CTAMGSPDC showed higher frequencies among others which correlating to higher affinity to amitrol and could be applied for bioreceptor research or gene engineering on bacteria as in previous study with metal ion binding sequences [9, 10]. Table 2. Amino acid sequences of constraint peptide having strong affinity to amitrol Amino acid sequence Frequency Amino acid sequence Frequency CSATPRPSC 13 CTLTGTHLC 2 CLIPTQAMC 12 CYTDTPTHC 1 CTAMGSPDC 10 CRNDLLNRC 1 CPNSAHANC 7 CGAGDKADC 1 CPVLYNSHC 5 CTQMTWSEC 1 CNMTQMKIC 5 CKNQWSISC 1 2010415no003:1_UV 2010415no003:1_Cond 2010415no003:1_Conc 0.0 2.0 4.0 6.0 8.0 mAu 0.0 10.0 20.0 30.0 40.0 50.0 60.0 70.0 80.0 ml Screening of peptide receptor sequences for amitrol detection 13 4. CONCLUSION Chromatographic biopanning was successfully used to screen for high affinity peptide sequences to amitrol, a hardly detected endocrine disrupting chemical. The combination of chromatographic biopanning, pH gradient washing buffer (pH 8- pH 2.2), and elution by sonication were applied during the screening. No phage was seen in washing buffer of second ground stated that these methods could shorten the number of biopanning. 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