Browse
Submit Data
Databases
API
Help

Dataset Information

0 Views

0 Connections

0 Citations

0 Reanalyses

0 Downloads

Omics score: 0

Phage display of functional, full-length human and viral membrane proteins.

ABSTRACT: Phage display of protein and peptide libraries offers a powerful technology for the selection and isolation of ligands and receptors. To date, the technique has been considered limited to soluble, non-membrane proteins. We report two examples of phage display of full-length, folded and functional membrane proteins. Consistent display required the recently reported KO7(+) helper phage. The two proteins, full-length caveolin-1 and HIV gp41, display well on the surface of the phage, and maintain their binding activities as shown by in vitro assays.

SUBMITTER: Majumdar S

PROVIDER: S-EPMC2597205 | biostudies-other | 2008 Nov

REPOSITORIES: biostudies-other

ACCESS DATA

Json Xml

Publications

Phage display of functional, full-length human and viral membrane proteins.

Majumdar Sudipta S Hajduczki Agnes A Mendez Aaron S AS Weiss Gregory A GA

Bioorganic & medicinal chemistry letters 20080717 22

Phage display of protein and peptide libraries offers a powerful technology for the selection and isolation of ligands and receptors. To date, the technique has been considered limited to soluble, non-membrane proteins. We report two examples of phage display of full-length, folded and functional membrane proteins. Consistent display required the recently reported KO7(+) helper phage. The two proteins, full-length caveolin-1 and HIV gp41, display well on the surface of the phage, and maintain th ...[more]

PMID: 18667306

Similar Datasets

The scope of phage display for membrane proteins.

Project description:Numerous examples of phage display applied to soluble proteins demonstrate the power of the technique for protein engineering, affinity reagent discovery and structure-function studies. Recent reports have expanded phage display to include membrane proteins (MPs). The scope and limitations of MP display remain undefined. Therefore, we report data from the phage display of representative types of membrane-associated proteins including plasma, nuclear, peripheral, single and multipass. The peripheral MP neuromodulin displays robustly with packaging by conventional M13-KO7 helper phage. The monotopic MP Nogo-66 can also display on the phage surface, if packaged by the modified M13-KO7(+) helper phage. The modified phage coat of KO7(+) can better mimic the zwitterionic character of the plasma membrane. Four examples of putatively ?-helical, integral MPs failed to express as fusions to an anchoring phage coat protein and therefore did not display on the phage surface. However, the ?-barrel MPs ShuA (Shigella heme uptake A) and MOMP (major outer membrane protein), which pass through the membrane 22 and 16 times, respectively, can display surprisingly well on the surfaces of both conventional and KO7(+) phages. The results provide a guide for protein engineering and large-scale mutagenesis enabled by the phage display of MPs.

| S-EPMC3230673 | biostudies-literature

Targeting membrane proteins for antibody discovery using phage display.

Project description:A critical factor in the successful isolation of new antibodies by phage display is the presentation of a correctly folded antigen. While this is relatively simple for soluble proteins which can be purified and immobilized onto a plastic surface, membrane proteins offer significant challenges for antibody discovery. Whole cell panning allows presentation of the membrane protein in its native conformation, but is complicated by a low target antigen density, high background of irrelevant antigens and non-specific binding of phage particles to cell surfaces. The method described here uses transient transfection of alternating host cell lines and stringent washing steps to address each of these limitations. The successful isolation of antibodies from a naive scFv library is described for three membrane bound proteins; human CD83, canine CD117 and bat CD11b.

| S-EPMC4870581 | biostudies-literature

Transpo-mAb display: Transposition-mediated B cell display and functional screening of full-length IgG antibody libraries.

Project description:In vitro antibody display and screening technologies geared toward the discovery and engineering of clinically applicable antibodies have evolved from screening artificial antibody formats, powered by microbial display technologies, to screening of natural, full-IgG molecules expressed in mammalian cells to readily yield lead antibodies with favorable properties in production and clinical applications. Here, we report the development and characterization of a novel, next-generation mammalian cell-based antibody display and screening platform called Transpo-mAb Display, offering straightforward and efficient generation of cellular libraries by using non-viral transposition technology to obtain stable antibody expression. Because Transpo-mAb Display uses DNA-transposable vectors with substantial cargo capacity, genomic antibody heavy chain expression constructs can be utilized that undergo the natural switch from membrane bound to secreted antibody expression in B cells by way of alternative splicing of Ig-heavy chain transcripts from the same genomic expression cassette. We demonstrate that stably transposed cells co-express transmembrane and secreted antibodies at levels comparable to those provided by dedicated constructs for secreted and membrane-associated IgGs. This unique feature expedites the screening and antibody characterization process by obviating the need for intermediate sequencing and re-cloning of individual antibody clones into separate expression vectors for functional screening purposes. In a series of proof-of-concept experiments, we demonstrate the seamless integration of antibody discovery with functional screening for various antibody properties, including binding affinity and suitability for preparation of antibody-drug conjugates.

| S-EPMC4966844 | biostudies-other

Nanodiscs allow phage display selection for ligands to non-linear epitopes on membrane proteins.

Project description:In this work, we exploited a method that uses polytopic membrane proteins as targets for phage display selections. Membrane proteins represent the largest class of drug targets and drug discovery is mostly based on the identification of ligands binding to target molecules. The screening of a phage display library for ligands against membrane proteins is typically hindered by the requirement of these proteins for a membrane environment, which is necessary to retain correct folding and epitope formation. Especially in proteins with multiple transmembrane domains, epitopes often are non-linear and consist of a combination of loops between transmembrane stretches of the proteins. Here, we have used bacteriorhodopsin (bR) as a model of polytopic membrane protein, assembled into nanoscale phospholipid bilayers, so called nanodiscs, to screen a phage display library for potential ligands. Nanodiscs provide a native-like environment to membrane proteins and thus selection of ligands can take place in a near physiological state. Screening a 12-mer phage display peptide library against bR nanodiscs led to the isolation of phage clones binding specifically to bR. We were further able to identify the binding site of selected phage clones proving that the clones bind to extramembranous, non-linear epitopes of bR. Thus, nanodiscs provide a suitable and general tool that allows screening of a phage display library against membrane proteins in a near native environment.

| S-EPMC3767683 | biostudies-literature

VirD: a virion display array for profiling functional membrane proteins.

Project description:To facilitate high-throughput biochemical analyses of membrane proteins, we have developed a novel display technology in a microarray format. Both single-pass (cluster of differentiation 4, CD4) and multiple-pass (G protein-coupled receptor 77, GPR77) human transmembrane proteins were engineered to be displayed in the membrane envelop of herpes simplex virions. These viruses produce large spherical virions displaying multiple copies of envelop proteins. Our aim was to engineer this virus to express these human proteins during the virus productive cycle and incorporate the human proteins into the virion during the assembly process. Another strategy presented includes engineering a fusion of glycoprotein C (gC), a major constituent of herpes simplex virus type 1 (HSV-1) virions, by hijacking the cis-acting signals to direct incorporation of the chimeric protein into the virion. The expression of the human proteins in infected cells, at the cell surface and in purified virions, is in the correct transmembrane orientation, and the proteins are biochemically functional. Purified virions printed on glass slides form a high-density Virion Display (VirD) Array, and the displayed proteins were demonstrated to retain their native conformations and interactions on the VirD Array judging by similar assays, such as antibody staining, as well as lectin and ligand binding. This method can be readily scaled or tailored for different modalities including a high-content, high-throughput platform for screening ligands and drugs of human membrane proteins.

| S-EPMC3791513 | biostudies-literature

Structural and Functional Properties of Viral Membrane Proteins

Project description:Viruses have developed a large variety of transmembrane proteins to carry out their infectious cycles. Some of these proteins are simply anchored to membrane via transmembrane helices. Others, however, adopt more interesting structures to perform tasks such as mediating membrane fusion and forming ion-permeating channels. Due to the dynamic or plastic nature shown by many of the viral membrane proteins, structural and mechanistic understanding of these proteins has lagged behind their counterparts in prokaryotes and eukaryotes. This chapter provides an overview of the use of NMR spectroscopy to unveil the transmembrane and membrane-proximal regions of viral membrane proteins, as well as their interactions with potential therapeutics.

| S-EPMC7122571 | biostudies-literature

Large-scale interaction profiling of PDZ domains through proteomic peptide-phage display using human and viral phage peptidomes.

Project description:The human proteome contains a plethora of short linear motifs (SLiMs) that serve as binding interfaces for modular protein domains. Such interactions are crucial for signaling and other cellular processes, but are difficult to detect because of their low to moderate affinities. Here we developed a dedicated approach, proteomic peptide-phage display (ProP-PD), to identify domain-SLiM interactions. Specifically, we generated phage libraries containing all human and viral C-terminal peptides using custom oligonucleotide microarrays. With these libraries we screened the nine PSD-95/Dlg/ZO-1 (PDZ) domains of human Densin-180, Erbin, Scribble, and Disks large homolog 1 for peptide ligands. We identified several known and putative interactions potentially relevant to cellular signaling pathways and confirmed interactions between full-length Scribble and the target proteins ?-PIX, plakophilin-4, and guanylate cyclase soluble subunit ?-2 using colocalization and coimmunoprecipitation experiments. The affinities of recombinant Scribble PDZ domains and the synthetic peptides representing the C termini of these proteins were in the 1- to 40-?M range. Furthermore, we identified several well-established host-virus protein-protein interactions, and confirmed that PDZ domains of Scribble interact with the C terminus of Tax-1 of human T-cell leukemia virus with micromolar affinity. Previously unknown putative viral protein ligands for the PDZ domains of Scribble and Erbin were also identified. Thus, we demonstrate that our ProP-PD libraries are useful tools for probing PDZ domain interactions. The method can be extended to interrogate all potential eukaryotic, bacterial, and viral SLiMs and we suggest it will be a highly valuable approach for studying cellular and pathogen-host protein-protein interactions.

| S-EPMC3932933 | biostudies-literature

Conformational Chaperones for Structural Studies of Membrane Proteins Using Antibody Phage Display with Nanodiscs.

Project description:A major challenge in membrane biophysics is to define the mechanistic linkages between a protein's conformational transitions and its function. We describe a novel approach to stabilize transient functional states of membrane proteins in native-like lipid environments allowing for their structural and biochemical characterization. This is accomplished by combining the power of antibody Fab-based phage display selection with the benefits of embedding membrane protein targets in lipid-filled nanodiscs. In addition to providing a stabilizing lipid environment, nanodiscs afford significant technical advantages over detergent-based formats. This enables the production of a rich pool of high-performance Fab binders that can be used as crystallization chaperones, as fiducial markers for single-particle cryoelectron microscopy, and as probes of different conformational states. Moreover, nanodisc-generated Fabs can be used to identify detergents that best mimic native membrane environments for use in biophysical studies.

| S-EPMC4740257 | biostudies-other

Recovering full-length viral genomes from metagenomes.

Project description:Infectious disease metagenomics is driven by the question: "what is causing the disease?" in contrast to classical metagenome studies which are guided by "what is out there?" In case of a novel virus, a first step to eventually establishing etiology can be to recover a full-length viral genome from a metagenomic sample. However, retrieval of a full-length genome of a divergent virus is technically challenging and can be time-consuming and costly. Here we discuss different assembly and fragment linkage strategies such as iterative assembly, motif searches, k-mer frequency profiling, coverage profile binning, and other strategies used to recover genomes of potential viral pathogens in a timely and cost-effective manner.

| S-EPMC4589665 | biostudies-literature

Cell surface display of functional human MHC class II proteins: yeast display versus insect cell display.

Project description:Reliable and robust systems for engineering functional major histocompatibility complex class II (MHCII) proteins have proved elusive. Availability of such systems would enable the engineering of peptide-MHCII (pMHCII) complexes for therapeutic and diagnostic applications. In this paper, we have developed a system based on insect cell surface display that allows functional expression of heterodimeric DR2 molecules with or without a covalently bound human myelin basic protein (MBP) peptide, which is amenable to directed evolution of DR2-MBP variants with improved T cell receptor (TCR)-binding affinity. This study represents the first example of functional display of human pMHCII complexes on insect cell surface. In the process of developing this pMHCII engineering system, we have also explored the potential of using yeast surface display for the same application. Our data suggest that yeast display is a useful system for analysis and engineering of peptide binding of MHCII proteins, but not suitable for directed evolution of pMHC complexes that bind with low affinity to self-reactive TCRs.

| S-EPMC3160208 | biostudies-literature

OmicsDI is part of the ELIXIR infrastructure

OmicsDI is an Elixir interoperability service. Learn more ›

Tweets

OmicsDI Databases

PRIDE
PeptideAtlas
MassIVE
JPOST Repository
Physiome Model Repository

EGA
EVA
ENA
LINCS
PAXDB
Cell Collective

MetaboLights
Metabolomics Workbench
MetabolomeExpress
GNPS
BioModels
FAIRDOMHub

ArrayExpress
dbGaP
ExpressionAtlas
GEO
NODE

Information

Databases
Help
API
Contact us
Code on GitHub
Terms of use
Submit Data