We describe a low cost, high throughput method to screen for fungal endoglucanase activity in E. coli. The method relies on a simple visual readout of substrate degradation, does not require enzyme purification, and is highly scalable. This allows for the rapid screening of large libraries of enzyme variants.
A procedure for liquid-based culturing and dispensing of C. elegans strains expressing fluorescent reporter proteins is described that does not require expensive sorting equipment. This approach can be applied to numerous inducible C. elegans genes for drug discovery or biosensing of contaminants.
We have developed a high-density microarray platform consisting of 3D nano-biofilms of C. albicans called CaBChip. The susceptibility profile of drugs tested on a CaBChip is comparable to the conventional 96-well plate model, suggesting that the fungal chip is ideally suited for true high-throughput screening of antifungal drugs.
High Throughput Quantitative Expression Screening and Purification Applied to Recombinant Disulfide-rich Venom Proteins Produced in E. coli
1Architecture et Fonction des Macromolécules Biologiques (AFMB), Aix-Marseille Université, 2iBiTec-S, Service d'Ingénierie Moléculaire des Protéines (SIMOPRO), Commissariat à l'énergie atomique et aux énergies alternatives (CEA) Saclay, France
A protocol for the quantitative, high throughput expression screening and analytical purification of fusion proteins from small-scale Escherichia coli cultures is described and applied to the expression of disulfide-rich animal venom protein targets.
Published July 30, 2014. Keywords: Bioengineering, E. coli, expression, recombinant, high throughput (HTP), purification, auto-induction, immobilized metal affinity chromatography (IMAC), tobacco etch virus protease (TEV) cleavage, disulfide bond isomerase C (DsbC) fusion, disulfide bonds, animal venom proteins/peptides
1Department of Pharmacology, Vanderbilt University School of Medicine, 2Department of Anesthesiology, Vanderbilt University School of Medicine, 3Vanderbilt Institute of Chemical Biology, Vanderbilt University School of Medicine
Methods for developing and validating a quantitative fluorescence assay for measuring the activity of inward rectifier potassium (Kir) channels for high-throughput compound screening is presented.
Published January 27, 2013. Keywords: Biochemistry, Molecular Biology, Chemistry, Cellular Biology, Chemical Biology, Pharmacology, Molecular Pharmacology, Potassium channels, drug discovery, drug screening, high throughput, small molecules, fluorescence, thallium flux, checkerboard analysis, DMSO, cell lines, screen, assay, assay development
1Unité de Génomique Virale et Vaccination, Virology Department, Institut Pasteur, CNRS UMR3569, 2Unité de Chimie et Biocatalyse, Biochemistry and Structural Biology Department, Institut Pasteur, CNRS UMR3523, 3Unité des Interactions Moléculaires Flavivirus-Hôtes, Virology Department, Institut Pasteur
In vitro assays to measure virus replication have been greatly improved by the development of recombinant RNA viruses expressing luciferase or other enzymes capable of bioluminescence. Here we detail a high-throughput screening pipeline that combines such recombinant strains of measles and chikungunya viruses to isolate broad-spectrum antivirals from chemical libraries.
We present a rapid and inexpensive screening method for identifying transcriptional regulators using high-throughput robotic transfections and a homemade dual-glow luciferase assay. This protocol rapidly generates direct side-by-side functional data for thousands of genes and is easily modifiable to target any gene of interest.
1Perkin Elmer Inc., 2Henry M. Jackson Foundation, 3The Geneva Foundation, 4ORISE, 5Frederick National Laboratory for Cancer Research, 6Division of Molecular and Translational Sciences, US Army Medical Research Institute of Infectious Diseases, 7DoD Biotechnology High Performance Computing Software Applications Institute (BHSAI), Telemedicine and Advanced Technology Research Center (TATRC), US Army Medical Research and Materiel Command (USAMRMC)
Botulinum neurotoxin is one of the most potent toxins among Category-A biothreat agents, yet a post-exposure therapeutic is not available. The high content imaging approach is a powerful methodology for identifying novel inhibitors as it enables multiparameter screening using biologically relevant motor neurons, the primary target of this toxin.
This article describes a novel protocol and reagent set designed for sensitive measurement of neurotoxic effects of compounds and treatments on co-cultures of neurons and astrocytes using high content analysis. Results demonstrate that high content analysis represents an exciting novel technology for neurotoxicity assessment.
Published May 5, 2009. Keywords: Neuroscience, high content screening, high content analysis, neurotoxicity, toxicity, drug discovery, neurite outgrowth, astrocytes, neurons, co-culture, immunofluorescence
Here we describe a novel high-content chemically induced inflammation assay aiming at the identification of immune-modulatory bioactives. We have successfully combined automated microscopy with custom developed software scripts enabling automated quantification of the inflammatory response as well as further data processing, analysis, mining, and storage.