Browse
Submit Data
Databases
API
Help

Dataset Information

9 Views

0 Connections

0 Citations

0 Reanalyses

0 Downloads

Omics score: 0

New platform for simple and rapid protein-based affinity reactions.

ABSTRACT: We developed a spongy-like porous polymer (spongy monolith) consisting of poly(ethylene-co-glycidyl methacrylate) with continuous macropores that allowed efficient in situ reaction between the epoxy groups and proteins of interest. Immobilization of protein A on the spongy monolith enabled high-yield collection of immunoglobulin G (IgG) from cell culture supernatant even at a high flow rate. In addition, immobilization of pepsin on the spongy monolith enabled efficient online digestion at a high flow rate.

SUBMITTER: Kubota K

PROVIDER: S-EPMC5428043 | biostudies-literature | 2017 Mar

REPOSITORIES: biostudies-literature

ACCESS DATA

Json Xml

Publications

New platform for simple and rapid protein-based affinity reactions.

Kubota Kei K Kubo Takuya T Tanigawa Tetsuya T Naito Toyohiro T Otsuka Koji K

Scientific reports 20170314 1

We developed a spongy-like porous polymer (spongy monolith) consisting of poly(ethylene-co-glycidyl methacrylate) with continuous macropores that allowed efficient in situ reaction between the epoxy groups and proteins of interest. Immobilization of protein A on the spongy monolith enabled high-yield collection of immunoglobulin G (IgG) from cell culture supernatant even at a high flow rate. In addition, immobilization of pepsin on the spongy monolith enabled efficient online digestion at a high ...[more]

PMID: 28282970

Similar Datasets

A protein functionalization platform based on selective reactions at methionine residues.

Project description:Nature has a remarkable ability to carry out site-selective post-translational modification of proteins, therefore enabling a marked increase in their functional diversity1. Inspired by this, chemical tools have been developed for the synthetic manipulation of protein structure and function, and have become essential to the continued advancement of chemical biology, molecular biology and medicine. However, the number of chemical transformations that are suitable for effective protein functionalization is limited, because the stringent demands inherent to biological systems preclude the applicability of many potential processes2. These chemical transformations often need to be selective at a single site on a protein, proceed with very fast reaction rates, operate under biologically ambient conditions and should provide homogeneous products with near-perfect conversion2-7. Although many bioconjugation methods exist at cysteine, lysine and tyrosine, a method targeting a less-explored amino acid would considerably expand the protein functionalization toolbox. Here we report the development of a multifaceted approach to protein functionalization based on chemoselective labelling at methionine residues. By exploiting the electrophilic reactivity of a bespoke hypervalent iodine reagent, the S-Me group in the side chain of methionine can be targeted. The bioconjugation reaction is fast, selective, operates at low-micromolar concentrations and is complementary to existing bioconjugation strategies. Moreover, it produces a protein conjugate that is itself a high-energy intermediate with reactive properties and can serve as a platform for the development of secondary, visible-light-mediated bioorthogonal protein functionalization processes. The merger of these approaches provides a versatile platform for the development of distinct transformations that deliver information-rich protein conjugates directly from the native biomacromolecules.

| S-EPMC6203954 | biostudies-literature

A Simple Platform for the Rapid Development of Antimicrobials.

Project description:Recent infectious outbreaks highlight the need for platform technologies that can be quickly deployed to develop therapeutics needed to contain the outbreak. We present a simple concept for rapid development of new antimicrobials. The goal was to produce in as little as one week thousands of doses of an intervention for a new pathogen. We tested the feasibility of a system based on antimicrobial synbodies. The system involves creating an array of 100 peptides that have been selected for broad capability to bind and/or kill viruses and bacteria. The peptides are pre-screened for low cell toxicity prior to large scale synthesis. Any pathogen is then assayed on the chip to find peptides that bind or kill it. Peptides are combined in pairs as synbodies and further screened for activity and toxicity. The lead synbody can be quickly produced in large scale, with completion of the entire process in one week.

| S-EPMC5730575 | biostudies-literature

A benchmarked protein microarray-based platform for the identification of novel low-affinity extracellular protein interactions.

Project description:Low-affinity extracellular protein interactions are critical for cellular recognition processes, but existing methods to detect them are limited in scale, making genome-wide interaction screens technically challenging. To address this, we report here the miniaturization of the AVEXIS (avidity-based extracellular interaction screen) assay by using protein microarray technology. To achieve this, we have developed protein tags and sample preparation methods that enable the parallel purification of hundreds of recombinant proteins expressed in mammalian cells. We benchmarked the protein microarray-based assay against a set of known quantified receptor-ligand pairs and show that it is sensitive enough to detect even very weak interactions that are typical of this class of interactions. The increase in scale enables interaction screening against a dilution series of immobilized proteins on the microarray enabling the observation of saturation binding behaviors to show interaction specificity and also the estimation of interaction affinities directly from the primary screen. These methodological improvements now permit screening for novel extracellular receptor-ligand interactions on a genome-wide scale.

| S-EPMC3325482 | biostudies-literature

MALDI-target integrated platform for affinity-captured protein digestion.

Project description:To address immunocapture of proteins in large cohorts of clinical samples high throughput sample processing is required. Here a method using the proteomic sample platform, ISET (integrated selective enrichment target) that integrates highly specific immunoaffinity capture of protein biomarker, digestion and sample cleanup with a direct interface to mass spectrometry is presented. The robustness of the on-ISET protein digestion protocol was validated by MALDI MS analysis of model proteins, ranging from 40 fmol to 1 pmol per nanovial. On-ISET digestion and MALDI MS/MS analysis of immunoaffinity captured disease-associated biomarker PSA (prostate specific antigen) from human seminal plasma are presented.

| S-EPMC3935078 | biostudies-literature

A new tool to guide halofunctionalization reactions: the halenium affinity (HalA) scale.

Project description:We introduce a previously unexplored parameter-halenium affinity (HalA)- as a quantitative descriptor of the bond strengths of various functional groups to halenium ions. The HalA scale ranks potential halenium ion acceptors based on their ability to stabilize a "free halenium ion". Alkenes in particular but other Lewis bases as well, such as amines, amides, carbonyls, and ether oxygen atoms, etc., have been classified on the HalA scale. This indirect approach enables a rapid and straightforward prediction of chemoselectivity for systems involved in halofunctionalization reactions that have multiple nucleophilic sites. The influences of subtle electronic and steric variations, as well as the less predictable anchimeric and stereoelectronic effects, are intrinsically accounted for by HalA computations, providing quantitative assessments beyond simple "chemical intuition". This combined theoretical-experimental approach offers an expeditious means of predicting and identifying unprecedented reactions.

| S-EPMC4183602 | biostudies-literature

RCADiA: simple automation platform for comparative multidimensional protein identification technology.

Project description:Multidimensional liquid chromatography in combination with tandem mass spectrometry has been used to analyze a variety of biological structures including protein complexes. Incorporating this approach with autosampling devices presents a number of problems including decreased sensitivity due to exposure to extra surfaces, carryover from run to run, and increased dead volume. We developed a device, termed Radial Column Array for Distribution and Automation (RCADiA), to automate multiple MuDPIT experiments while eliminating many of these problems and maintaining a high resolution and sensitive analysis. The design, which places each sample downstream of any common fluid path, presents a low risk of carryover between successive analyses. Beyond the convenience of automation, the RCADiA platform also produces data of similar quality to the standard method of performing individual MuDPIT experiments. We demonstrate this device by performing a comparative analysis of mitochondria enriched from rat liver and spinal cord.

| S-EPMC2528021 | biostudies-literature

Affinity capture of polyribosomes followed by RNAseq (ACAPseq), a discovery platform for protein-protein interactions

Project description:Defining protein-protein interactions (PPIs) is central to the biological sciences. Here, we present a novel platform - Affinity Capture of Polyribosomes followed by RNA sequencing (ACAPseq) - for identifying PPIs. ACAPseq harnesses the power of massively parallel RNA sequencing (RNAseq) to quantify the enrichment of polyribosomes based on the affinity of their associated nascent polypeptides for an immobilized protein “bait”. This method was developed and tested using neonatal mouse brain polyribosomes and a variety of extracellular domains as baits. Of 92 baits tested, 25 identified one or more binding partners that appear to be biologically relevant; additional candidate partners remain to be validated. ACAPseq can detect binding to targets that are present at less than 1 part in 100,000 in the starting polyribosome preparation. One of the observed PPIs wes analyzed in detail, revealing the mode of homophilic binding for Protocadherin-9 (PCDH9), a non-clustered Protocadherin family member.

2018-10-20 | GSE121524 | GEO

Rapid Protein-Ligand Affinity Determination by Photoinduced Hyperpolarized NMR.

Project description:The binding affinity determination of protein-ligand complexes is a cornerstone of drug design. State-of-the-art techniques are limited by lengthy and expensive processes. Building upon our recently introduced novel screening method utilizing photochemically induced dynamic nuclear polarization (photo-CIDNP) NMR, we provide the methodological framework to determine binding affinities within 5-15 min using 0.1 mg of protein. The accuracy of our method is demonstrated for the affinity constants of peptides binding to a PDZ domain and fragment ligands binding to the protein PIN1. The method can also be extended to measure the affinity of nonphoto-CIDNP-polarizable ligands in competition binding experiments. Finally, we demonstrate a strong correlation between the ligand-reduced signals in photo-CIDNP-based NMR fragment screening and the well-established saturation transfer difference (STD) NMR. Thus, our methodology measures protein-ligand affinities in the micro- to millimolar range in only a few minutes and informs on the binding epitope in a single-scan experiment, opening new avenues for early stage drug discovery approaches.

| S-EPMC11228983 | biostudies-literature

Immuno Affinity SELEX for Simple, Rapid, and Cost-Effective Aptamer Enrichment and Identification against Aflatoxin B1.

Project description:Aflatoxins are naturally occurring mycotoxins that contaminate food and agro commodities, leading to acute and chronic health conditions in human and animals. In the present work, an attempt was made to generate high-affinity single stranded DNA aptamers that specifically bind to Aflatoxin B1 (AFB1) by a modified Systemic Evolution of Ligands by Exponential Enrichment (SELEX) procedure with the aid of Immunoaffinity columns. Ten rounds of SELEX and alternating three counter SELEX rounds with a cocktail of related and other mycotoxins were performed to enhance the specificity. Resultant 105 aptamers were clustered into 12 groups according to their primary sequence homology. Candidates with lowest Gibbs free energy (dG value) and unique stem loop structures were selected for further characterization. Aptamers, AFLA5, AFLA53, and AFLA71 exhibiting lower Kd values (50.45 ± 11.06, 48.29 ± 9.45, and 85.02 ± 25.74 nM) were chosen for development of ELONA and determination of purification ability of toxin. The detection limit (LOD) of AFLA5 and AFLA71 was 20 and 40 ng/ml, respectively. HPLC analysis implied that selected aptamers were able to recover and quantify 82.2 to 96.21% (LOQ - 53.74 ng) and 78.3 to 94.22% (LOQ - 66.75 ng) of AFB1 from spiked corn samples, respectively. These findings indicate, immunoaffinity based SELEX can pave an alternative approach to screen aptamers against mycotoxin detection and purification.

| S-EPMC5130984 | biostudies-literature

Performance of HADDOCK and a simple contact-based protein-ligand binding affinity predictor in the D3R Grand Challenge 2.

Project description:We present the performance of HADDOCK, our information-driven docking software, in the second edition of the D3R Grand Challenge. In this blind experiment, participants were requested to predict the structures and binding affinities of complexes between the Farnesoid X nuclear receptor and 102 different ligands. The models obtained in Stage1 with HADDOCK and ligand-specific protocol show an average ligand RMSD of 5.1 Å from the crystal structure. Only 6/35 targets were within 2.5 Å RMSD from the reference, which prompted us to investigate the limiting factors and revise our protocol for Stage2. The choice of the receptor conformation appeared to have the strongest influence on the results. Our Stage2 models were of higher quality (13 out of 35 were within 2.5 Å), with an average RMSD of 4.1 Å. The docking protocol was applied to all 102 ligands to generate poses for binding affinity prediction. We developed a modified version of our contact-based binding affinity predictor PRODIGY, using the number of interatomic contacts classified by their type and the intermolecular electrostatic energy. This simple structure-based binding affinity predictor shows a Kendall's Tau correlation of 0.37 in ranking the ligands (7th best out of 77 methods, 5th/25 groups). Those results were obtained from the average prediction over the top10 poses, irrespective of their similarity/correctness, underscoring the robustness of our simple predictor. This results in an enrichment factor of 2.5 compared to a random predictor for ranking ligands within the top 25%, making it a promising approach to identify lead compounds in virtual screening.

| S-EPMC5767195 | 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