Isolation of Erythropoietin Receptor Agonist Peptides Using Evolved Phage Libraries

McConnell, Stephen J.; Dinh, Thai; Le, Mong-Huong; Brown, Steven J; Becherer, Kathleen; Blumeyer, Kirsten; Kautzer, Curtis; Axelrod, Fumiko; Spinella, Dominic G.

doi:10.1515/bchm.1998.379.10.1279

Cited by 37 publications

(21 citation statements)

References 15 publications

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“…The greater flexibility regarding the residues that can be randomized provides a larger interaction surface between the binding partners during the binding event. The discovery of an agonist in the 3F library also demonstrates the utility of this approach, particularly from the viewpoint that agonists have been identified from immunoglobulin sources [32-34] and random peptide libraries [35,36], but there are no published reports of agonistic molecules from non-immunoglobulin proteins [9]. The high affinity and remarkable specificity of 3F proteins for their targets, along with their stability, make this scaffold suitable for diagnostic, therapeutic and protein chip purposes.…”

Section: Discussionmentioning

confidence: 99%

Directed evolution of a three-finger neurotoxin by using cDNA display yields antagonists as well as agonists of interleukin-6 receptor signaling

et al. 2011

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BackgroundDirected evolution of biomolecules such as DNA, RNA and proteins containing high diversity has emerged as an effective method to obtain molecules for various purposes. In the recent past, proteins from non-immunoglobulins have attracted attention as they mimic antibodies with respect to binding potential and provide further potential advantages. In this regard, we have attempted to explore a three-finger neurotoxin protein (3F). 3F proteins are small (~7 kDa), structurally well defined, thermally stable and resistant to proteolysis that presents them as promising candidates for directed evolution.ResultsWe have engineered a snake α-neurotoxin that belongs to the 3F family by randomizing the residues in the loops involved in binding with acetylcholine receptors and employing cDNA display to obtain modulators of interleukin-6 receptor (IL-6R). Selected candidates were highly specific for IL-6R with dissociation constants and IC50s in the nanomolar range. Antagonists as well as agonists were identified in an IL-6 dependent cell proliferation assay. Size minimization yielded peptides of about one-third the molecular mass of the original proteins, without significant loss of activities and, additionally, lead to the identification of the loops responsible for function.ConclusionsThis study shows 3F protein is amenable to introduce amino acid changes in the loops that enable preparation of a high diversity library that can be utilized to obtain ligands against macromolecules. We believe this is the first report of protein engineering to convert a neurotoxin to receptor ligands other than the parent receptor, the identification of an agonist from non-immunoglobulin proteins, the construction of peptide mimic of IL-6, and the successful size reduction of a single-chain protein.

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Section: Discussionmentioning

confidence: 99%

Directed evolution of a three-finger neurotoxin by using cDNA display yields antagonists as well as agonists of interleukin-6 receptor signaling

et al. 2011

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“…antibody) from a phage display library. Magnetic particles which, compared to immunotubes or microtitre plate wells, have the advantage of much higher binding capacities, have been the preferred solid phase for this kind of phage display selection [2,[31][32][33][34][35][36][37].…”

Section: Cdna Expression Phage Librariesmentioning

confidence: 99%

High-throughput Screening of Surface Displayed Gene Products

Walter¹,

Konthur²,

Lehrach³

2012

CCHTS

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Abstract:With the human genome project approaching completion, there is a growing interest in functional analysis of gene products. The characterization of large numbers of proteins, their expression patterns and in vivo localisations, demands the use of automated technology that maintains a logistic link to the encoding genes. As a complementary approach, phage display is used for recombinant protein expression and the selection of interacting (binding) molecules. Cloning of libraries in filamentous bacteriophage or phagemid vectors provides a physical link between the expressed protein and its encoding DNA sequence. High-throughput technology for automated library handling and phage display selection has been developed using picking-spotting robots and a module for pin-based magnetic particle handling. This system enables simultaneous interaction screening of libraries and the selection of binders to different target molecules at high throughput. Target molecules are either displayed on high-density filter membranes (protein filters) or tag-bound to magnetic particles and can be handled as native ligands. Binding activity is confirmed by magnetic particle ELISA in the microtitre format. The whole procedure from immobilisation of target molecules to confirmed clones of binders is automatable. Using this technology, we have selected human scFv antibody fragments against expression products of human cDNA libraries.

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“…The technology, often requiring creative modifications, was used for epitope mapping of antibodies [114], screening for receptor agonists and antagonists [115,133,134], in vitro evolution of antibodies [116,117] and antibody surrogates in the form of randomized fragments on diverse scaffold proteins [118,119], discovery of enzyme substrates [120,121] and inhibitors [113,122,135], identifying functionally coupled proteins and analysis of protein-protein interactions [123,124], designing catalytic antibodies (abzymes) and enzymes with novel specificities [125], improving proteolytic and folding stability of muteins [126], vaccine design [131,132], and construction of gene delivery vehicles [127]. Protein ligands selected from phage libraries as well as whole recombinant phage have been used as affinity matrices in chromatographic techniques [128,129] and biosensing [130].…”

Section: Recent Exciting Applications Of Phage Display Technologymentioning

confidence: 99%

Progress in phage display: evolution of the technique and its applications

Bratkovič

2009

Cell. Mol. Life Sci.

176

117

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Phage display, the presentation of (poly)peptides as fusions to capsid proteins on the surface of bacterial viruses, celebrates its 25th birthday in 2010. The technique, coupled with in vitro selection, enables rapid identification and optimization of proteins based on their structural or functional properties. In the last two decades, it has advanced tremendously and has become widely accepted by the scientific community. This by no means exhaustive review aims to inform the reader of the key modifications in phage display. Novel display formats, innovative library designs and screening strategies are discussed. I will also briefly review some recent uses of the technology to illustrate its incredible versatility.

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Isolation of Erythropoietin Receptor Agonist Peptides Using Evolved Phage Libraries

Cited by 37 publications

References 15 publications

Directed evolution of a three-finger neurotoxin by using cDNA display yields antagonists as well as agonists of interleukin-6 receptor signaling

Directed evolution of a three-finger neurotoxin by using cDNA display yields antagonists as well as agonists of interleukin-6 receptor signaling

High-throughput Screening of Surface Displayed Gene Products

Progress in phage display: evolution of the technique and its applications

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