Biochemistry

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    Protocols in Current Issue
    Production and Crystallization of Nanobodies in Complex with the Receptor Binding Domain of the SARS-CoV-2 Spike Protein
    [Abstract]

    The receptor binding domain (RBD) of the spike protein of SARS-CoV-2 binds angiotensin converting enzyme-2 (ACE-2) on the surface of epithelial cells, leading to fusion, and entry of the virus into the cell. This interaction can be blocked by the binding of llama-derived nanobodies (VHHs) to the RBD, leading to virus neutralisation. Structural

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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: 551, 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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    Novel Protein Production Method Combining Native Expression in Human Cells with an Intein-based Affinity Purification and Self-cleavable Tag
    Authors:  Peter J. Carman and Roberto Dominguez, date: 03/20/2022, view: 462, Q&A: 0
    [Abstract]

    The human proteins used in most biochemical studies are commonly obtained using bacterial expression. Owing to its relative simplicity and low cost, this approach has been extremely successful, but is inadequate for many proteins that require the mammalian folding machinery and posttranslational modifications (PTMs) for function. Moreover, the

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    Heterologous Expression and High Degree Purification of the Restriction Endonuclease SauUSI
    Authors:  Vinayak Sadasivam Tumuluri and Kayarat Saikrishnan, date: 01/05/2022, view: 1544, Q&A: 0
    [Abstract]

    Mechanisms that target and destroy foreign nucleic acids are major barriers to horizontal gene transfer (HGT) in prokaryotes. Amongst them, restriction-modification (R-M) systems are found in ≥75% of the sequenced genomes in Bacteria and Archaea. Due to their high target sequence specificity and potent nucleolytic activity, R-M systems are used

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    Transient yet Robust Expression of Proteins in the Mouse Liver via Intravenous Injection of Lipid Nanoparticle-encapsulated Nucleoside-modified mRNA
    [Abstract]

    With the recent availability of the SARS-CoV-2 mRNA-based vaccines, public attention has been drawn to this new technology and how it may be applied to other indications. Temporal activation of key hepatic regenerative pathways can induce liver regeneration, overcoming the lack of donor organs for liver transplantation and ineffectiveness of

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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: 1510, 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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    Cell-free Translation: Preparation and Validation of Translation-competent Extracts from Saccharomyces cerevisiae
    Authors:  Brandon M. Trainor, Anton A. Komar, Dimitri G. Pestov and Natalia Shcherbik, date: 09/20/2021, view: 1647, Q&A: 0
    [Abstract]

    Cell-free translation is a powerful technique for in vitro protein synthesis. While cell-free translation platforms prepared from bacterial, plant, and mammalian cells are commercially available, yeast-based translation systems remain proprietary knowledge of individual labs. Here, we provide a detailed protocol for simple, fast, and

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    Implementing Novel Designs in pET Expression Plasmids that Increase Protein Production
    Authors:  Patrick J. Shilling and Daniel O. Daley, date: 08/20/2021, view: 4998, Q&A: 0
    [Abstract]

    pET expression plasmids are widely used in the biotechnology, biopharmaceutical, and basic research sectors for the production of recombinant proteins. Typically, they are used off-the-shelf because they support high production titers; however, we have identified two design flaws in many pET plasmids that limit their production capacity. We used

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    One-step White Blood Cell Extracellular Staining Method for Flow Cytometry
    [Abstract]

    Flow cytometry is a powerful analytical technique that is increasingly used in scientific investigations and healthcare; however, it requires time-consuming, multi-step sample procedures, which limits its use to specialized laboratories. In this study, we propose a new universal one-step method in which white blood cell staining and red blood cell

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    Cell-free Synthesis of Correctly Folded Proteins with Multiple Disulphide Bonds: Production of Fungal Hydrophobins
    Authors:  Rezwan Siddiquee and Ann H Kwan, date: 05/20/2021, view: 3235, Q&A: 0
    [Abstract]

    Cell-free synthesis is a powerful technique that uses the transcriptional and translational machinery extracted from cells to create proteins without the constraints of living cells. Here, we report a cell-free protein production protocol using Escherichia coli lysate (Figure 1) to successfully express a class of proteins (known as hydrophobins)

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