Biopanning Strategy Research Articles

504. Neuronal Affinity of a C7C Loop Peptide Identified through Phage Display

Background: Phage display is a promising tool for the screening of peptides with high affinity for specific cells. Here we describe a novel peptide with neuronal affinity isolated from a C7C library. The C7C library is based on a randomized 7-mer sequence flanked by two cysteine residues. In contrast to linear peptides displayed in other libraries, these cysteine residues form a disulfide cross-link, resulting in a loop structure.Methods: We designed a two-tiered biopanning strategy initially selecting for ganglioside (GT1b) binding and subsequently selecting for binding to NGF-differentiated pheochromocytoma (PC12) cells. We next evaluated the binding characteristics of the predominant clones by the bound phage ratio and by immunofluorescence phage localization. Finally, the predominant clone was synthesized and fluorescein conjugated, in order to further assess its neuronal binding.Results: Sequencing random phage plaques revealed amplification of a dominant clone, TetC7C.1 (54.8%). Immunofluorescence binding studies using phage antibodies to detect the bound phage on the surface of the cells revealed selective binding of this clone to NGF-differentiated PC12 cells. Moreover, the synthetic peptide demonstrated specific binding to neuronal cell lines (SH-SY5Y, NSC-34 and NGF-differentiated PC12 cells) and neuronal tissue (DRG and spinal cord).Conclusions: The C7C structure creates a loop that minimizes the impact of peptide insertion on the confirmation of the recipient protein. Loop peptides are preferable for the modification of proteins through insertion at sites other than the terminal regions. Furthermore, small loop peptides have the ideal characteristics for modification of viral vector capsids without undermining genome packaging. Our biopanning strategy favored the selection of a peptide with enhanced binding for the clostridial tetanus toxin receptor GT1b. The neuronal binding properties of the Tet.C7C.1 peptide may be applied in the development of neurotropic viral vectors and therapeutic fusion proteins with enhanced binding to axon terminals. Background: Phage display is a promising tool for the screening of peptides with high affinity for specific cells. Here we describe a novel peptide with neuronal affinity isolated from a C7C library. The C7C library is based on a randomized 7-mer sequence flanked by two cysteine residues. In contrast to linear peptides displayed in other libraries, these cysteine residues form a disulfide cross-link, resulting in a loop structure. Methods: We designed a two-tiered biopanning strategy initially selecting for ganglioside (GT1b) binding and subsequently selecting for binding to NGF-differentiated pheochromocytoma (PC12) cells. We next evaluated the binding characteristics of the predominant clones by the bound phage ratio and by immunofluorescence phage localization. Finally, the predominant clone was synthesized and fluorescein conjugated, in order to further assess its neuronal binding. Results: Sequencing random phage plaques revealed amplification of a dominant clone, TetC7C.1 (54.8%). Immunofluorescence binding studies using phage antibodies to detect the bound phage on the surface of the cells revealed selective binding of this clone to NGF-differentiated PC12 cells. Moreover, the synthetic peptide demonstrated specific binding to neuronal cell lines (SH-SY5Y, NSC-34 and NGF-differentiated PC12 cells) and neuronal tissue (DRG and spinal cord). Conclusions: The C7C structure creates a loop that minimizes the impact of peptide insertion on the confirmation of the recipient protein. Loop peptides are preferable for the modification of proteins through insertion at sites other than the terminal regions. Furthermore, small loop peptides have the ideal characteristics for modification of viral vector capsids without undermining genome packaging. Our biopanning strategy favored the selection of a peptide with enhanced binding for the clostridial tetanus toxin receptor GT1b. The neuronal binding properties of the Tet.C7C.1 peptide may be applied in the development of neurotropic viral vectors and therapeutic fusion proteins with enhanced binding to axon terminals.

915. High and Low Affinity Consensus Binding Sequences to Extracellular α7 Integrin X2B Subunit Detected Using Phage Display Biopanning

Multiple biopanning strategies have been developed to uncover ligands that bind to specific cellular receptors such as panning on immobilized targets or on live cells. This study utilized a cell-based system in which MCF7 breast carcinoma cells stably transfected with the |[alpha]|7 integrin X2B subunit gene (MCF7|[alpha]|7(+)) were used for positive selection, while non-transfected MCF7 cells were used for negative selection. We chose |[alpha]|7 integrin, a cell surface receptor whose expression is largely restricted to skeletal muscle and is up-regulated in dystrophic muscle, as our biopanning receptor to discover targeting ligands for muscle gene delivery. The biopanning strategy developed for this project began with a single library of phage representing approximately 1|[times]|109 possible combinations of 7 amino acids. For the first round of biopanning, we incubated the original library with MCF7|[alpha]|7(+) cells for positive selection, removed any non-binding phage and collected the bound phage in 2 separate fractions. A low affinity fraction was collected by low pH wash and then neutralized, while a high affinity fraction was recovered by cell collection, subsequent freezing and thawing to lyse the cells, and removal of cellular debris by centrifugation. Once the positive selection was completed, we immediately performed negative selection using each fraction (low and high affinity) separately on MCF7 cells. After the last of 6 rounds of biopanning for each phage fraction, with each round containing a positive and negative selection, the un-amplified eluate was titered to limiting dilution and individual phage clones were picked and amplified separately. Purified clones were analyzed first by an ELISA-based binding assay developed. using MCF7|[alpha]|7(+) and MCF7 cells to determine if a single phage clone with specific ligand revealed higher binding specificity in |[alpha]|7- positive cells. Following binding analysis, DNA from clones with 2-3|[times]| higher binding affinity in |[alpha]|7-positive cells over |[alpha]|7-negative cells were sequenced to determine if a consensus binding sequence had been discovered. We show that 36% and 70% of analyzed clones from low and high affinity binding fractions, respectively, revealed a consensus binding sequence to |[alpha]|7 integrin. Further testing of each consensus sequence will be conducted to determine efficiency and specificity of muscle binding. The sequences can then be incorporated into viral and non-viral vectors for vector targeting.

Neuronal affinity of a C7C loop peptide identified through phage display

Phage display is a promising tool for the screening of peptides with high affinity for specific cells. Here we describe a novel peptide with neuronal affinity isolated from a C7C library. We designed a two-tiered biopanning strategy initially selecting for ganglioside binding and subsequently selecting for binding to PC12 cells. At the completion of biopanning, 54.8% of phage clones bore the identical peptide (Tet.C7C.1). Immunofluorescence confirmed selective binding of this clone to differentiated PC12 cells. Tet.C7C.1 was synthesized and fluorescein conjugated. The synthetic peptide binds neuronal cell lines (SH-SY5Y, NSC-34 and PC12 cells) and tissue (DRG and spinal cord). The C7C structure creates a loop that minimizes the impact of peptide insertion on the confirmation of the recipient protein. Small loop peptides have the ideal characteristics for modification of viral vector capsids without undermining genome packaging. The neuronal binding properties of this peptide may be applied in the development of neurotropic viral vectors.

855. In Vivo Spinal Cord Uptake and Neuronal Binding Properties of Tet1: A Potential Means for Enhanced Spinal Cord AAV Delivery

Background: On the basis of its extremely efficient uptake and delivery to motor neurons, the tetanus toxin might be useful for neurospecific binding. However, the immune response to the toxin due the vaccination against tetanus, represents an obstacle to its clinical applicability. In order to find specific peptide sequences that can mimic binding of C fragment, we isolated a short neurotropic peptide sequence which when inserted into the AAV vector's coat might retarget the vector toward neuronal membranes without hindering its transduction efficiency. We have previously reported the phage display biopanning strategy for isolation of peptides with specific affinity for the trisialoganglioside (GT1b) Clostridial toxin receptor1 This process identified Tet1, a 12 AA peptide with specific and enhanced binding to differentiated pheochromocytoma (PC12) cells, primary motor neurons, and dorsal root ganglion (DRG) cells in vitro. The present experiment assessed the receptor binding characteristics of this peptide as well as its uptake and delivery to spinal motor neurons in vivo.

439. Neuronal Membrane Specific Binding Peptides Isolated Via Phage Display Biopanning Against Trisialogangliosides

Background: The tropism of adeno-associated viral vectors can be altered by inserting peptides into the vector's coat. However, large peptides may alter the capacity of VP proteins to package DNA. In order to enhance the neural affinity and specificity of AAV binding, we have attempted to isolate short peptides which when inserted into the cap gene of the AAV genome, would retarget the vector toward neuronal membranes without hindering its transduction efficiency. We have previously reported a phage display biopanning strategy for the isolation of such peptides (Liu et al Mol Ther: 7(5) S351, 2003). We used trisialogangliosides (GT1b), a known receptor of Clostridial tetanus toxin present on neuronal membranes, as our target. Tetanus toxin owes its high neuronal affinity to specific GT1b binding providing an ideal interaction to replicate in AAV. Using a random 12 amino acid peptide phage library, we conducted a novel four round biopanning process against immobilized GT1b. In the final three rounds of biopanning, we eluted with recombinant Tetanus Toxin C Fragment (rTTC) to recover phage that specifically mimic its binding to GT1b. Four candidate peptides were enriched following the biopanning process. We selected the most common peptide (Clone 2A) which was present in 70% of the sequenced phage clones along with a second peptide (Clone 2B) that shared similar homology with Clone 2A (Liu et al Mol Ther: 7(5) S351, 2003).

Biopanning phage display libraries using magnetic beads vs. polystyrene plates.

BioTechniquesVol. 26, No. 2 BenchmarksOpen AccessBiopanning Phage Display Libraries Using Magnetic Beads Vs. Polystyrene PlatesStephen J. McConnell, Thai Dinh, Mong-Huong Le & Dominic G. SpinellaStephen J. McConnell*Address correspondence to Dr. Stephen J. McConnell, Chugai Biopharmaceuticals, 6275 Nancy Ridge Drive, San Diego, CA 92121, USA. Internet: E-mail Address: smcconnell@chugaibio.comChugai Biopharmaceuticals, San Diego, CA, USASearch for more papers by this author, Thai DinhChugai Biopharmaceuticals, San Diego, CA, USASearch for more papers by this author, Mong-Huong LeChugai Biopharmaceuticals, San Diego, CA, USASearch for more papers by this author & Dominic G. SpinellaChugai Biopharmaceuticals, San Diego, CA, USASearch for more papers by this authorPublished Online:22 Aug 2018https://doi.org/10.2144/99262bm06AboutSectionsPDF/EPUB ToolsAdd to favoritesDownload CitationsTrack Citations ShareShare onFacebookTwitterLinkedInRedditEmail FiguresReferencesRelatedDetailsCited ByGigavalent Display of Proteins on Monodisperse Polyacrylamide Hydrogels as a Versatile Modular Platform for Functional Assays and Protein Engineering1 August 2022 | ACS Central Science, Vol. 8, No. 8Evaluation of Phage Display Biopanning Strategies for the Selection of Anti-Cell Surface Receptor Antibodies30 July 2022 | International Journal of Molecular Sciences, Vol. 23, No. 15Emerging ELISA derived technologies for in vitro diagnosticsTrAC Trends in Analytical Chemistry, Vol. 152Auto-Panning: a highly integrated and automated biopanning platform for peptide screening1 January 2021 | Lab on a Chip, Vol. 21, No. 14Development of phage biopanning strategies to identify affinity peptide ligands for kappa light chain Fab fragments25 July 2019 | Biotechnology Progress, Vol. 35, No. 6Phage display antibody libraries: A robust approach for generation of recombinant human monoclonal antibodiesInternational Journal of Biological Macromolecules, Vol. 135Invert biopanning: A novel method for efficient and rapid isolation of scFvs by phage display technologyBiologicals, Vol. 44, No. 6Continuous microfluidic assortment of interactive ligands (CMAIL)31 August 2016 | Scientific Reports, Vol. 6, No. 1Calf thymus histone-conjugated magnetic poly(2-oxoethyl methacrylate) microspheres for affinity isolation of anti-histone IgGs from the blood serum of patients with systemic lupus erythematosus1 January 2015 | RSC Advances, Vol. 5, No. 77Affinity Selection of Peptide Binders with Magnetic Beads via Organic Phase Separation (MOPS)Biological & Pharmaceutical Bulletin, Vol. 38, No. 11Micro/Nano Technologies and Their Biological and Medical ApplicationsPhage ESCape: An emulsion-based approach for the selection of recombinant phage display antibodiesJournal of Immunological Methods, Vol. 367, No. 1-2Generation of Fab Fragment-Like Molecular Recognition Proteins against Staphylococcal Enterotoxin B by Phage Display TechnologyClinical and Vaccine Immunology, Vol. 17, No. 11Generation of a Human Anti-Tumor Necrosis Factor-α Monoclonal Antibody by in Vitro Immunization with a Multiple Antigen Peptide22 May 2014 | Bioscience, Biotechnology, and Biochemistry, Vol. 74, No. 9Automation in the High-throughput Selection of Random Combinatorial Libraries—Different Approaches for Select Applications8 April 2010 | Molecules, Vol. 15, No. 4A Novel Strategy for Proteome-wide Ligand Screening Using Cross-linked Phage MatricesJournal of Biological Chemistry, Vol. 285, No. 13Production of Human Monoclonal Antibodies against FcεRIα by a Method Combining in Vitro Immunization with Phage Display22 May 2014 | Bioscience, Biotechnology, and Biochemistry, Vol. 73, No. 7Automated Panning and Screening Procedure on Microplates for Antibody Generation from Phage Display LibrariesSLAS Discovery, Vol. 14, No. 3A rapid and efficient strategy to generate antigen-specific human monoclonal antibody by in vitro immunization and the phage display methodJournal of Immunological Methods, Vol. 332, No. 1-2Magnetic moment measurement of magnetic nanoparticles using atomic force microscopy17 December 2007 | Measurement Science and Technology, Vol. 19, No. 1Addressing the Protein Crystallization Bottleneck By Cocrystallization23 October 2007 | Crystal Growth & Design, Vol. 7, No. 11Templated synthesis of gold–iron alloy nanoparticles using pulsed laser deposition28 September 2006 | Nanotechnology, Vol. 17, No. 20Synthesis and Characterization of Paramagnetic Microparticles through Emulsion-Templated Free Radical Polymerization16 February 2006 | Langmuir, Vol. 22, No. 6Phage-display selection of antibodies to the left end of CTX3C using synthetic fragmentsJournal of Immunological Methods, Vol. 289, No. 1-2Phage display for epitope determination: A paradigm for identifying receptor–ligand interactionsMagnetic-bead enzyme-linked immunosorbent assay verifies adsorption of ligand and epitope accessibilityAnalytical Biochemistry, Vol. 311, No. 2Automation of phage display for high-throughput antibody developmentTARGETS, Vol. 1, No. 1Phage display combinatorial libraries of short peptides: ligand selection for protein purificationEnzyme and Microbial Technology, Vol. 28, No. 9-10Bead-based HTS applications in drug discoveryDrug Discovery Today, Vol. 5 Vol. 26, No. 2 Follow us on social media for the latest updates Metrics Downloaded 1,416 times History Published online 22 August 2018 Published in print February 1999 Information© 2018 Author(s)PDF download