Rounds Of Biopanning Research Articles

Binding of the Receptor Tyrosine Kinase TrkB to the Neural Cell Adhesion Molecule (NCAM) Regulates Phosphorylation of NCAM and NCAM-dependent Neurite Outgrowth

Recognition molecules and neurotrophins play important roles during development and maintenance of nervous system functions. In this study, we provide evidence that the neural cell adhesion molecule (NCAM) and the neurotrophin receptor TrkB directly interact via sequences in their intracellular domains. Stimulation of TrkB by brain-derived neurotrophic factor leads to tyrosine phosphorylation of NCAM at position 734. Mutation of this tyrosine to phenylalanine completely abolishes tyrosine phosphorylation of NCAM by TrkB. Moreover, the knockdown of TrkB in hippocampal neurons leads to a reduction of NCAM-induced neurite outgrowth. Transfection of NCAM-deficient hippocampal neurons with mutated NCAM carrying an exchange of tyrosine by phenylalanine at position 734 leads to promotion of NCAM-induced neurite outgrowth in comparison with that observed after transfection with wild-type NCAM, whereas a reduction of neurite outgrowth was observed after transfection with mutated NCAM, which carries an exchange of tyrosine by glutamate that mimics the phosphorylated tyrosine. Our observations indicate a functional relationship between TrkB and NCAM.

Bioscreening of phage display antibody library and expression of a humanized single‐chain variable fragment antibody against human connective tissue growth factor (CTGF/CCN2)

Excessive expression of CTGF (connective tissue growth factor)/CCN2 has been observed in many fibrotic diseases. The inhibition of the CTGF/CCN2 by antibody has been shown to be clinically useful for the management of fibrosis. A phage display humanized single-chain Fv antibody library was screened using CTGF/C (CTGF/CCN2 C-terminal domain) as the target. A phage ELISA was performed after four rounds of biopanning, and ten positive clones were further evaluated by ELISA and were chosen for DNA sequencing. The DNA encoding scFv (single-chain variable fragment) containing a full-length variable domain fragment of heavy chain and light chain of human immunoglobulin was inserted into pET-32(a)+ vector, and the fusion protein (TrxA-scFv) containing a thrombin cleavage site was expressed mainly in soluble form. The scFv was obtained by purified fusion protein digested with thrombin and then separated from the fusion partner TrxA by gel-filtration chromatography. An immunological assay showed that the purified scFv reacted with CTGF/CCN2 in a concentration-dependent manner. The result of the cell migration assay demonstrated that the scFv at 100 ng/ml could effectively inhibit the migration of HUVEC (human umbilical-vein endothelial cells) caused by CTGF/C. The number of migratory cells was significantly decreased as compared with the negative control (1062+/-92 versus 3269+/-288, P<0.001) and the inhibition rate was 90.5%.

Abstract 343: High-throughput screening of multiple protein kinase inhibitor using polymeric nanoparticles

Abstract Protein kinase play pivotal regulatory roles in most cell communication and metabolic pathways. Inhibitors of protein kinases not only hold great promise as therapeutic agents for many diseases, especially cancer, but are also of profound utility in the characterization of signaling pathways because of the centrality of protein phosphorylation as a regulatory process. Therefore, sensitive and widely applicable detection of protein kinase activity will provide a valuable tool to screen protein kinase inhibitors in drug discovery. We previously reported that self-assembled poly-ion complexes (PICs) containing kemptide, protein kinase A specific peptide motif, recognize the phosphorylation of kemptide by protein kinase A by recovery of fluorescence. In this research we found kinase specific substrate peptide with phage display which an efficient primary screening method to detect protein-protein interaction because of its relatively easy, rapid and massive recovery and analysis. Kinase substrate peptide library was made with phage and then library is displayed on capsid protein of phage. After kinase reaction, the phage clones are selected, recovered, and amplified for next round of bio-panning. The candidate clones are selected according for at least 4th round of bio-panning and the resultant peptides are analyzed for specificity. To prepare PICs, found peptide was conjugated with polyehtyleneimine (PEI) labeled with cyanine 5.5 (cy5.5). PICs consisting of peptide and cy5.5 chemically conjugated PEI and poly-L-aspartic acid showed significant fluorescent signal recovery in presence of protein kinase while they hardly showed fluorescent signal in absence of protein kinase. In addition, fluorescent signal did not recover with protein kinase inhibitor even though protein kinase was present. We tried to apply highthroughput screening to perform chemical library screening with 384-well plate. While wells without protein kinase showed strong fluorescent signal, wells containing protein kinase inhibitors represented no fluorescent signal, which is fully consistent with previous results and our expects. We conclude that this system is applied to highthroughput screening systems targeting protein kinase inhibitors. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 343.

Abstract 1625: Discovery of a breast cancer targeting peptide with anti-tumor activity

Abstract The American Cancer Society estimates 192,370 women will be diagnosed with breast cancer in the United States in 2009, and approximately 40,170 will die of this disease. Towards identifying new ligand-directed therapies for breast cancer, we have developed an in vivo screening method, where peptides that home specifically to the vascular component of tissues are selected after intravenous administration of an engineered combinatorial peptide phage library. We have used this technology for the identification of a new breast cancer targeting peptide and its receptor in a mouse model of highly aggressive and vascularized breast cancer. A random phage library displaying the cyclic sequence CX7C (X = any of the 20 natural amino acids) was injected intravenously in tumor-bearing mice after which phage was recovered from the tumor-tissue by bacterial infection and re-injected for a subsequent round of biopanning. After sequencing of the recovered phage from round 2 and 3 several candidate peptides were identified. One breast cancer targeting peptide (BCTP) was recovered from all tumors with the highest frequency. This peptide showed marked tumor homing as verified by immunohistochemistry (IHC). We used peptide affinity chromatography and tandem mass spectrometry (MS/MS) to identify the target receptor of BCTP. Strikingly, this newly discovered receptor plays an as yet unrecognized role in the pathology of breast cancer. BCPT exhibited strong binding to its receptor as verified by ELISA. To investigate if the BCTP receptor is a relevant candidate drug target in breast cancer, we obtained biopsy samples of breast cancer patients prior to chemotherapy. Strong immunoreactivity of the BCTP receptor was observed in the paraffin embedded tissue sections. Next, mammary tumor-bearing mice were injected twice per week with BCTP, control peptide or saline, systemically. BCTP treated mice demonstrated a marked decrease in tumor size of treated mice, compared to mice injected with PBS alone. We have identified, characterized and validated the peptide BCTP, specifically homing to highly aggressive, vascularized breast tumors. In addition to its tumor targeting capabilities this peptide possesses anti-tumor function in itself, inhibiting tumor growth and progression in tumor-bearing mice. More importantly, the target receptor expression has been verified in human biopsy samples of breast cancer, indicating that this protein might be a promising new drug target. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 1625.

Terpestacin Inhibits Tumor Angiogenesis by Targeting UQCRB of Mitochondrial Complex III and Suppressing Hypoxia-induced Reactive Oxygen Species Production and Cellular Oxygen Sensing

Cellular oxygen sensing is required for hypoxia-inducible factor-1alpha stabilization, which is important for tumor cell survival, proliferation, and angiogenesis. Here we find that terpestacin, a small molecule previously identified in a screen of microbial extracts, binds to the 13.4-kDa subunit (UQCRB) of mitochondrial Complex III, resulting in inhibition of hypoxia-induced reactive oxygen species generation. Consequently, such inhibition blocks hypoxia-inducible factor activation and tumor angiogenesis in vivo, without inhibiting mitochondrial respiration. Overexpression of UQCRB or its suppression using RNA interference demonstrates that it plays a crucial role in the oxygen sensing mechanism that regulates responses to hypoxia. These findings provide a novel molecular basis of terpestacin targeting UQCRB of Complex III in selective suppression of tumor progression.

Screening and evaluating the mimic peptides as a useful serum biomarker of ankylosing spondylitis using a phage display technique

To screen specific serum biomarker for ankylosing spondylitis (AS) using a phage random peptide library. A phage random peptide library of random peptide 12-mers was immunoscreened with purified immunoglobulin (Ig) G from sera of AS patients. Positive clones obtained after three rounds of biopanning were detected with ELISA and sequenced. Reaction of the screened positive clones with sera from AS patients, systemic lupus erythematosus (SLE) patients, rheumatoid arthritis (RA) patients, osteoarthritis (OA) patients and healthy controls was detected using phage ELISA. Correlation among erythrocyte sedimentation rate (ESR), C-reactive protein (CRP), and the absorbance value of the positive clone in phage ELISA was examined in AS patients. Seventeen out of twenty randomly selected phage clones exhibited specific reaction with purified sera IgG from AS patients, among them seven coming from the same clone whose inserted peptide sequence was LALPPLAPNHHH (named "AS1"). Phage ELISA results showed that the positive reaction rate of the AS1 clone was 92.0% with AS patients, significantly different (P<0.01) from those with SLE patients (56.7%), RA patients (50.0%), OA patients (13.3%), and healthy controls (14.0%). Absorbance value of the AS1 clone in phage ELISA was significantly higher than those in the other groups (P<0.05). In addition, the absorbance value of the AS1 clone showed no statistically significant correlation with ESR and CRP in AS patients, suggesting that AS1 detects AS patients through a unique mechanism other than inflammation. The short peptide AS1 obtained through screening of a phage random peptide library with purified serum IgG from AS patients can specifically react with the sera of AS patients, and thereby may be a candidate of AS-specific serum biomarkers.

Phage display peptide probes for imaging early response to bevacizumab treatment

Early evaluation of cancer response to a therapeutic regimen can help increase the effectiveness of treatment schemes and, by enabling early termination of ineffective treatments, minimize toxicity, and reduce expenses. Biomarkers that provide early indication of tumor therapy response are urgently needed. Solid tumors require blood vessels for growth, and new anti-angiogenic agents can act by preventing the development of a suitable blood supply to sustain tumor growth. The purpose of this study is to develop a class of novel molecular imaging probes that will predict tumor early response to an anti-angiogenic regimen with the humanized vascular endothelial growth factor antibody bevacizumab. Using a bevacizumab-sensitive LS174T colorectal cancer model and a 12-mer bacteriophage (phage) display peptide library, a bevacizumab-responsive peptide (BRP) was identified after six rounds of biopanning and tested in vitro and in vivo. This 12-mer peptide was metabolically stable and had low toxicity to both endothelial cells and tumor cells. Near-infrared dye IRDye800-labeled BRP phage showed strong binding to bevacizumab-treated tumors, but not to untreated control LS174T tumors. In addition, both IRDye800- and (18)F-labeled BRP peptide had significantly higher uptake in tumors treated with bevacizumab than in controls treated with phosphate-buffered saline. Ex vivo histopathology confirmed the specificity of the BRP peptide to bevacizumab-treated tumor vasculature. In summary, a novel 12-mer peptide BRP selected using phage display techniques allowed non-invasive visualization of early responses to anti-angiogenic treatment. Suitably labeled BRP peptide may be potentially useful pre-clinically and clinically for monitoring treatment response.

Biopanning of HCV antigen epitopes from a random 12-peptide library by anti-hepatitis C virus polyclonal antibody

To analyze the epitopes of anti-hepatitis C virus(HCV)antibodies by peptide library biopanning. Phage random peptide library of 12 amino acids was immunoscreened with purified IgG from the sera of hepatitis C patients. Positive clones which were obtained after 3 rounds of biopanning were detected by ELISA and 4 of them were sequenced. After 3 rounds of screening, the radio of output to input increased from (4.6 x 10(-4))% to (5.3 x 10(-2))%, meaning the enrichment was effective. At the third round of screening, all the selected clones proved to specifically react with the sera for immunoscreening. Four positive phage clones were sequenced, which shared a very conservative sequence and was named as C1. Its inserting sequence in the coat protein III was deduced to be GSMSPYVRWYTP, and the positive rate of C1 reacted with 20 cases of HCV patients was 85%. The antigen-mimic peptide C1 is successfully screened from 12 random phage peptide library and it has some diagnostic value.

Selection of peptides bind to PreS1 antigen from a phage-displayed peptide library

Objective:To screen for peptides that specifically bind to PreS1 antigen from a phage-displayed peptide library.Methods: The PreS1 antigen was used as the target molecule to screen the binding peptide from the Ph.D.-7 peptide library with phage display technique,and the positive clones were identified by ELISA.Results: After three rounds of biopanning,the binding peptides were screened from the peptide library by ELISA and competitive inhibiting ELISA.Sequencing result showed that the binding peptides had high affinity and specificity.Conclusion: A peptide binding PreS1 antigen has been successfully obtained by screening the phage display library,which paves a way for the diagnosis and treatment of hepatitis B infection.

Peptide aptamers against titanium-based implants identified through phage display

Commercially pure titanium (cp-Ti) is widely used in the field of long-term clinical oral implantology owing to its ability to allow close bone-implant apposition. The optimization of its function based on artificial proteins has become a key issue in the development of improved cp-Ti implants. Here, we set out to identify peptide aptamers with preferential adsorption towards titanium-based implants through the phage display methodology. Fifteen sequences were selected in the third round of biopanning. One sequence, ATWVSPY (named TBP1), had a 40% repetition rate and exhibited the strongest binding affinity to cp-Ti disks. Ten sequences were selected in the fourth round, among which the repetition rate is 80% for TBP1 and 20% for TBP2 (GVGLPHT). The peptide aptamers against cp-Ti disks can provide an alternative method of functional coating for biomaterial surfaces.

Recombinant Peptides as Biomarkers for Tumor Response to Molecular Targeted Therapy

Phage display technology can be used to identify peptide sequences that bind rapidly and specifically to tumors responding to sunitinib therapy. These peptides may help to address problems with current methods of assessing tumor response to therapy that can be slow and have limited usage. The peptide of interest was isolated after four rounds of biopanning in MDA-MB-231 and MCF-7 xenografted tumors. The binding location of the peptide was investigated with immunohistochemistry. Its in vivo ability to bind to breast tumors responding to therapy was determined by treating nude mice, xenografted with various tumor cell lines, with sunitinib and using near IR imaging to assess the ability of the peptide conjugated to Alexafluor-750 to bind tumors. EGEVGLG was the dominant sequence isolated from biopanning. This peptide showed increased binding relative to control groups in two cancer cell lines (MDA-MB-435 and MCF-7 human breast) responding to sunitinib treatment, whereas no elevated binding occurred in vitro when samples were incubated with tumor cells that are unresponsive to sunitinib treatment (B16 melanoma and BxPC3 pancreatic). Mice xenografted with tumors that are responsive to sunitinib therapy showed increased peptide binding when compared with untreated control. Mice bearing tumors unresponsive to sunitinib therapy showed no increased peptide binding between treated and untreated groups. The use of recombinant peptides to assess the pharmacodynamic response of cancer holds promise in minimizing the duration of ineffective treatment regimens in patients, potentially providing a more rapid and less invasive assessment of cancer response to systemic therapy.

Magnetic Resonance Molecular Imaging of Vascular Cell Adhesion Molecule-1 Expression in Inflammatory Lesions Using a Peptide-Vectorized Paramagnetic Imaging Probe

The vascular cell adhesion molecule-1 (VCAM-1) has distinct roles in inflammatory cell recruitment to the damaged vessel wall. In the present work, a cyclic heptapeptide phage displayed library was screened in vitro during four rounds of biopanning. On the basis of Kd and IC50 values, a peptide (encoded as R832) was selected for in vitro and in vivo validation. After conjugation to Gd-DOTA, VCAM-1 imaging was assessed by MRI on a model of T cell mediated hepatitis, induced in mice by concanavalin A. On histological samples, the location of biotinylated R832 (R832-Bt) around liver veins in hepatitis resembles the pattern of MRI enhancement. Gd-DOTA-R832 was then assessed on ApoE(-/-) mice and produced an important signal enhancement of the aortic wall, while R832-Bt interacted with morphologic structures comparable to those marked by anti-VCAM-1 antibody. In conclusion, the in vitro and in vivo evaluation of peptide R832 suggests a specific interaction with the targeted biomolecule. Its conjugation to imaging reporters could assist the diagnosis of inflammatory diseases.

Peptide mimotopes of rabies virus glycoprotein with immunogenic activity

A random constrained hexapeptide phage display library (Cys-6aa-Cys) was screened with purified neutralizing human anti-rabies virus IgG antibodies (hRABVIgG) to identify peptides that correspond to or mimic natural epitopes on rabies virus glycoprotein (RABVG) and to investigate their immunogenicities in vivo. After four rounds of biopanning, 20 phage clones randomly selected for their specificity to hRABVIgG, effectively blocked the binding of the inactive rabies virus (RABV) to hRABVIgG. The phage clones were sequenced and the deduced amino acid sequences were derived (C-KRDSTW-C; C-KYLWSK-C; C-KYWLSR-C; C-KYWWSK-C; C-KYAWSR-C; C-KYSMSK-C). Alignments to the amino acid sequence of RABVG showed good match with the antigenic site III (at 330–338 aa), indicating that the hRABVIgG antibodies most likely recognize preferentially this antigenic site. The selected mimotopes were able to inhibit the interactions of the hRABVIgG antibodies with RABV in a dose-dependent manner. Subcutaneous administration of phageKRDSTW expressing the RABVG site III mimotope induced an RABVG-specific IgG response in BALB/c mice. The results indicated that peptide mimotopes when displayed on phages, are accessible to the mice immune system to trigger a humoral response and to induce IgG production. The RABVG site III mimotope (C-KRDSTW-C) would provide a new and promising concept for the development of rabies vaccine.

Screening for peptides binding on Phytophthora capsici extracts by phage display

Phytophthora blight of pepper is a completely destructive plant disease caused by the oomycete pathogen Phytophthora capsici Leonian. Phytophthora disease is responsible for major losses in pepper production and the pathogen can survive in soil in the absence of the host plant for many years. Currently, there are no early diagnostic reagents available that specifically target P. capsici. Therefore, diagnostic tools that can detect Phytophthora are required. In the present study, we screened for P. capsici-binding peptides using M13 phage display. After five rounds of biopanning, we identified P. capsici-binding peptides from a random peptide library that showed high binding affinity and specificity toward P. capsici in the picomolar range. These peptides can be used to develop novel diagnostic probes or potent inhibitors with diverse polyvalencies.

Discovery and Characterization of a Peptide Motif That Specifically Recognizes a Non-native Conformation of Human IgG Induced by Acidic pH Conditions

In therapeutic antibody preparation, acidic pH conditions are generally used for elution from Protein A affinity column of IgG or for its viral inactivation. Exposing IgG to low pH conditions induces conformational changes, leading to its functional damage or loss, although the mechanisms have not been fully elucidated. In this study using random peptide T7 phage display libraries, we isolated a unique and novel peptide motif that specifically recognized the non-native conformer (acid conformer) of human IgG that was generated by the low pH treatment, but not the native conformer. We examined the generation conditions and biochemical properties of acid conformer using the peptide motif as an affinity ligand. The acid conformer was easily generated at acidic pH (<pH 3.0) and at moderate temperatures (20-40 degrees C). The conformer was present in a monomeric form functionally maintaining antigen or Fc receptor binding, but showed a tendency to aggregate with a long incubation time at neutral pH (>25 degrees C). The peptides isolated here could contribute to the elucidation of the mechanisms of antibody dysfunction or aggregation during acid exposure as well as storage of human IgG.

Identification of biologically active peptides that inhibit binding of human CXCL8 to its receptors from a random phage-epitope library

A random bacteriophage peptide library was used to map structural features of human (h)CXCR1 and hCXCR2 by determining the epitopes of neutralizing mAb 5A12 anti-hCXCR1 and mAb 6C6 anti-hCXCR2. After three rounds of biopanning, five mAb5 A12- and four mAb 6C6-binding peptides were identified from a 6-mer peptide library. Consensus sequences (S/T)(1)(F/A/N/D)(2)(I/M)(3)W(4)D(5)F(6) and F/L/M)(1)W(2)(D/N/L)(3)D(4)F(5)W(6) were deduced from sequences of these peptides. They correspond to a highly conserved N-domain sequence (9)MWDF(12) of hCXCR1 and (13)DFW(15) of hCXCR2. The phage bearing the peptides showed specific binding to immobilized mAb 5A12 or mAb 6CC, and over 86% of phages bound were competitively inhibited by free synthetic peptides. In FACScan analysis, all selected phage peptides were able to strongly inhibit the binding of mAb 5A12 and mAb 6C6 to hCXCR1- and hCXCR2-transfected preB 300-19 murine cells. Furthermore, synthetic peptides of the corresponding phage epitopes were effective in blocking the antibody-CXCR1/2 interactions and to inhibit the binding of hCXCL8 to hCXCR1 and hCXCR2 transfectants. Peptides 5A12/2 (SAMWDF) and 6C6/1 (FWDDFW) competed effectively for (125)I-hCXCL8 binding to hCXCR1 and hCXCR2 with IC(50), respectively, equal to 10 muM and 5.4 muM. Calcium release and chemotaxis of hCXCR1/2 transfectants or human neutrophils were inhibited by all peptides in a dose-dependent manner. Furthermore, the peptide 6C6/1 FWDDFW showed inhibitory effects on chemotaxis of human netrophils induced by hCXCR2 chemokines such as hCXCL1-3 and hCXCL5. Specificities of peptides 5A12/2 and 6C6/1 were assessed with hCXCR3, hCXCR4, hCXCR5, hCCR3, and hCCR5 receptors. In vivo, peptides 5A12/2 and p6C6/1 blockade hCXCL8-induced neutrophil recruitment in skin inflammation in rabbits. Taken together, these data demonstrate that phage-display analysis provides information about the relative location of amino acids on the N-domain surfaces of hCXCR1 and hCXCR2 proteins using antibody imprints of the receptor-surface structure. The derived mimotopes could be used as inhibitors of hCXCL8-induced activities related to its interaction with the N-domain of hCXCR1 and hCXCR2.

Klebsiella pneumoniae MrkD adhesin-mediated immunity to respiratory infection and mapping the antigenic epitope by phage display library

In this study, the K. pneumoniae MrkD adhesin was identified an immunodominant antigen which correlated with protection against infection by K. pneumoniae. The mouse monoclonal antibodies (mAbs) against MrkD adhesin were produced by the hybridoma technique using recombinant MrkD-GST as the immunogen and were immunoscreened against phage-displayed random dodecapeptide library (Ph.D.-12). After three rounds of biopanning, 36 phage clones were randomly selected and their specificity to mAb was verified by sandwich and competitive inhibition ELISA. Sixteen phage clones were sequenced and their amino acids were deduced. One mimotope (QKTLAKSTYMSA) showed good match with MrkD adhesion at 148–159 aa and the serum of mice induced by the phage clone clearly recognized MrkD adhesion.

A novel affinity ligand for polystyrene surface from a phage display random library and its application in anti-HIV-1 ELISA system

In order to develop an affinity ligand for site-directed immobilization of target proteins on polystyrene (PS) surface, a linear 12-mer peptide phage display random library was screened. Phage clones that specifically bound to PS plate were sequenced after three rounds of biopanning. The obtained DNA sequences revealed that there were several aromatic and basic amino acid residues, which may be critical to binding. One of the selected dodecapeptides, named Lig1 (FKFWLYEHVIRG), was genetically fused to the N/C-terminus of recombinant antigen ENV which could be recognized by specific antibodies against human immunodeficiency virus type 1 (HIV-1), to investigate its performance as an affinity ligand. The ligand-fused ENVs overexpressed in Escherichia coli were compared to the original one in terms of the immobilization characteristics on PS plate in enzyme-linked immunosorbent assay (ELISA). The results indicated that the ligand-fused proteins showed a considerably improved affinity to PS surface, and were preferentially adsorbed on PS plate suffering only scarcely from interference by coexisting protein molecules. Anti-HIV-1 ELISA system, which employed Lig1- ENV (Lig1 fused to ENV N-terminus) as immobilization antigen also exhibited sufficiently high sensitivity and specificity in serodiagnosis tests.

Identification of Burkholderia pseudomallei Mimotope using Phage Display Approach

This study reportsa sequential biopanning against serum obtained from a patient infected with B. pseudomallei using a phage display peptide library. A peptide bearing the consensus sequence NKNSFDAWLQSF was obtained after 3 rounds of biopanning and it partly resembles the sequence 35FDAWLAAQEF44 of B. pseudomallei site-specific recombinase, which is a phage integrase family protein. Overall, the mimotope obtained in this study could be a potential candidate for the development of a cost effective diagnostic test or vaccine in the future.

Generation and Validation of a Shewanella oneidensis MR-1 Clone Set for Protein Expression and Phage Display

A comprehensive gene collection for S. oneidensis was constructed using the lambda recombinase (Gateway) cloning system. A total of 3584 individual ORFs (85%) have been successfully cloned into the entry plasmids. To validate the use of the clone set, three sets of ORFs were examined within three different destination vectors constructed in this study. Success rates for heterologous protein expression of S. oneidensis His- or His/GST- tagged proteins in E. coli were approximately 70%. The ArcA and NarP transcription factor proteins were tested in an in vitro binding assay to demonstrate that functional proteins can be successfully produced using the clone set. Further functional validation of the clone set was obtained from phage display experiments in which a phage encoding thioredoxin was successfully isolated from a pool of 80 different clones after three rounds of biopanning using immobilized anti-thioredoxin antibody as a target. This clone set complements existing genomic (e.g., whole-genome microarray) and other proteomic tools (e.g., mass spectrometry-based proteomic analysis), and facilitates a wide variety of integrated studies, including protein expression, purification, and functional analyses of proteins both in vivo and in vitro.