Biochemistry

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    Protocols in Current Issue
    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: 63, 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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    Methods for the Extraction of Heme Prosthetic Groups from Hemoproteins
    Authors:  Kat Ellis-Guardiola, Jess Soule and Robert T. Clubb, date: 09/20/2021, view: 102, Q&A: 0
    [Abstract]

    Hemoproteins are widely researched because they contain redox-active heme prosthetic groups (iron + protoporphyrin IX) that enable them to perform a range of vital functions, acting as enzymes, participants in electron transfer reactions, or gas sensing, carrying, and storage proteins. While the heme prosthetic group is almost always essential for

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    Solubilization Method for Isolation of Photosynthetic Mega- and Super-complexes from Conifer Thylakoids
    Authors:  Pushan Bag, Wolfgang P. Schröder, Stefan Jansson and Domenica Farci, date: 09/05/2021, view: 478, Q&A: 0
    [Abstract]

    Photosynthesis is the main process by which sunlight is harvested and converted into chemical energy and has been a focal point of fundamental research in plant biology for decades. In higher plants, the process takes place in the thylakoid membranes where the two photosystems (PSI and PSII) are located. In the past few decades, the evolution of

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    Purification of Mitochondrial Ribosomes with the Translocase Oxa1L from HEK Cells
    Authors:  Hanting Yang and Nirupa Desai, date: 08/05/2021, view: 985, Q&A: 0
    [Abstract]

    Mitochondrial ribosomes (mitoribosomes) perform protein synthesis inside mitochondria, the organelles responsible for energy conversion and adenosine triphosphate (ATP) production in eukaryotic cells. To investigate their functions and structures, large-scale purification of intact mitoribosomes from mitochondria-rich animal tissues or HEK cells

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    Purification of Recombinant Wild Type and Mutant Ryanodine Receptors Expressed in HEK293 Cells
    [Abstract]

    High quantities of purified ryanodine receptor (RyR), a large (2.26 MDa) intracellular homotetrameric membrane protein, can be obtained from heterologous expression in HEK293 cells and used for structure determination by cryo-EM. The advantage of using recombinant protein is that the variability due to post-translational modifications can be

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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: 2351, 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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    Expression and Purification of the Human Cation-chloride Cotransporter KCC1 from HEK293F Cells for Structural Studies
    Authors:  Si Liu and Jiangtao Guo, date: 04/05/2021, view: 789, Q&A: 0
    [Abstract]

    Cation-chloride cotransporters (CCCs) mediate the coupled, electroneutral symport of cations such as Na+ and/or K+ with chloride across membrane. Among CCCs family, K-Cl cotransporters (KCC1-KCC4) extrude intracellular Cl- by the transmembrane K+ gradient. In humans, these KCCs play vital roles in the physiology of the nervous system and kidney.

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    Expression and Purification of Yeast-derived GPCR, Gα and Gβγ Subunits for Structural and Dynamic Studies
    Authors:  Wenjie Zhao, Xudong Wang and Libin Ye, date: 02/20/2021, view: 3550, Q&A: 0
    [Abstract]

    In the last several years, as evidence of a surged number of GPCR-G complex structures, the expressions of GPCRs and G proteins for structural biology have achieved tremendous successes, mostly in insect and mammalian cell systems, resulting in more than 370 structures of over 70 GPCRs have been resolved. However, the challenge remains,

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    Purification of Cytosolic Phospholipase A2α C2-domain after Expression in Soluble Form in Escherichia coli
    [Abstract]

    Previous expression/purification strategies for cytosolic phospholipase A2α C2-domain in Escherichia coli have relied on refolded protein recovered from inclusion bodies and sometimes containing C-terminal Cys139Ala and Cys141Ser substitutions to eliminate potential refolding complications induced by Cys residues. The protocol presented

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    Reverse Phase-high-performance Liquid Chromatography (RP-HPLC) Analysis of Globin Chains from Human Erythroid Cells
    Authors:  Anne Chalumeau, Giacomo Frati, Elisa Magrin and Annarita Miccio, date: 01/20/2021, view: 1290, Q&A: 0
    [Abstract]

    β-hemoglobinopathies are severe genetic disorders characterized either by the abnormal synthesis of the adult β-globin chains of the hemoglobin (Hb) tetramer (βS-globin chains) in sickle cell disease (SCD) or by the reduced β-globin production in β-thalassemia. The identification and quantification of globin chains are

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