Prokaryotes

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    Protocols in Current Issue
    Purification of the Bacterial Amyloid “Curli” from Salmonella enterica Serovar Typhimurium and Detection of Curli from Infected Host Tissues
    [Abstract]

    Microbiologists are learning to appreciate the importance of “functional amyloids” that are produced by numerous bacterial species and have impacts beyond the microbial world. These structures are used by bacteria to link together, presumably to increase survival, protect against harsh conditions, and perhaps to influence cell-cell

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    Expression, Purification, and in vitro Enzyme Activity Assay of a Recombinant Aldehyde Dehydrogenase from Thermus thermophilus, using an Escherichia coli host
    Authors:  Kim Shortall, Edmond Magner and Tewfik Soulimane, date: 05/05/2022, view: 555, Q&A: 0
    [Abstract]

    Based on previous in-depth characterisation, aldehyde dehydrogenases (ALDH) are a diverse superfamily of enzymes, in terms of both structure and function, present in all kingdoms of life. They catalyse the oxidation of an aldehyde to carboxylic acid using the cofactor nicotinamide adenine dinucleotide (phosphate) (NAD(P)+), and are often not

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    Simple Scalable Protein Expression and Extraction Using Two-stage Autoinducible Cell Autolysis and DNA/RNA Autohydrolysis in Escherichia coli
    Authors:  Romel Menacho-Melgar and Michael D. Lynch, date: 01/20/2022, view: 1011, Q&A: 0
    [Abstract]

    Recombinant protein expression is extensively used in biological research. Despite this, current protein expression and extraction methods are not readily scalable or amenable for high-throughput applications. Optimization of protein expression conditions using traditional methods, reliant on growth-associated induction, is non-trivial. Similarly,

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    Urea Denaturation, Zinc Binding, and DNA Binding Assays of Mutant p53 DNA-binding Domains and Full-length Proteins
    Authors:  Jeung-Hoi Ha, Xin Yu, Darren R. Carpizo and Stewart N. Loh, date: 10/20/2021, view: 927, Q&A: 0
    [Abstract]

    In the cell, the thermodynamic stability of a protein – and hence its biological activity – can change dramatically as a result of perturbations in its amino acid sequence and the concentration of stabilizing ligands. This interplay is particularly evident in zinc-binding transcription factors such as the p53 tumor suppressor, whose

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    Biophysical Characterization of Iron-Sulfur Proteins
    Authors:  Bhanu P. Jagilinki, Irina Paluy, Alexei M. Tyryshkin, Vikas Nanda and Dror Noy, date: 10/20/2021, view: 1084, Q&A: 0
    [Abstract]

    Iron-sulfur proteins are primordial catalysts and biological electron carriers that today drive major metabolic pathways across all forms of life. They can access a diversity of oxidation states and can mediate electron transfer over an extended range of reduction potentials spanning more than 1 V. Depending on the protein micro-environment and

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    Anaerobic Expression and Purification of Holo-CCIS, an Artificial Iron-sulfur Protein
    Authors:  Bhanu P. Jagilinki, Irina Paluy, Vikas Nanda and Dror Noy, date: 09/20/2021, view: 1512, Q&A: 0
    [Abstract]

    Iron-sulfur proteins are ubiquitous among all living organisms and are indispensable for almost all metabolic pathways ranging from photosynthesis, respiration, nitrogen, and carbon dioxide cycles. The iron-sulfur clusters primarily serve as electron acceptors and donors and transfer electrons to active sites of various enzymes, thus driving the

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    A Co-purification Method for Efficient Production and Src Kinase-mediated Phosphorylation of Aplysia Cortactin
    Authors:  Sherlene L. Brown, Yuan Ren, Daniel M. Suter and Seema Mattoo, date: 09/20/2021, view: 1240, Q&A: 0
    [Abstract]

    Cortactin is an actin-binding protein that regulates processes like cell migration, endocytosis, and tumor cell metastasis. Although cortactin is associated with actin-cytoskeletal dynamics in non-neuronal cells and cell-free systems, the exact mechanisms underlying its fundamental roles in neuronal growth cones are not fully explored. Recent

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    Preparation and Characterization of Internally Modified DNA Templates for Chemical Transcription Roadblocking
    Author:  Eric J. Strobel, date: 09/05/2021, view: 1119, Q&A: 0
    [Abstract]

    Site-specific transcription arrest is the basis of emerging technologies that assess nascent RNA structure and function. Cotranscriptionally folded RNA can be displayed from an arrested RNA polymerase (RNAP) for biochemical manipulations by halting transcription elongation at a defined DNA template position. Most transcription

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    Monitoring Protein Splicing Using In-gel Fluorescence Immediately Following SDS-PAGE
    Authors:  Joel Weinberger II and Christopher W. Lennon, date: 08/20/2021, view: 2087, Q&A: 0
    [Abstract]

    Inteins garner significant interest from both basic and applied researchers due to their unique catalytic abilities. Herein, we describe a protocol for accurately monitoring protein splicing without purification using in-gel fluorescence immediately following Tris-Glycine SDS-PAGE. Following expression in Escherichia coli, cells are lysed by

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    Synchronized Real-time Measurement of Sec-mediated Protein Translocation
    Authors:  Riti Gupta, Dmitri Toptygin and Christian M. Kaiser, date: 08/20/2021, view: 1400, Q&A: 0
    [Abstract]

    The Sec translocon, consisting of a heterotrimeric transmembrane channel (SecYEG) and an associated ATPase (SecA), catalyzes the export of unfolded proteins from the cytosol in bacteria. Kinetically resolving protein translocation at high resolution yields mechanistic insight into the process. Translocation is typically followed by measuring the

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