Biochemistry

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    Protocols in Current Issue
    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: 2081, 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: 592, 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: 3258, 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: 1119, 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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    Preparation of Bacterial Outer Membrane Vesicles for Characterisation of Periplasmic Proteins in Their Native Environment
    Authors:  Johannes Thoma and Björn M. Burmann, date: 12/20/2020, view: 1484, Q&A: 0
    [Abstract]

    Bacterial outer membrane vesicles (OMVs) are naturally formed by budding from the outer membrane of Gram-negative bacteria. OMVs consist of a lipid bilayer identical in composition to the original outer membrane and contain periplasmic content within their lumen. Enriched with specific envelope proteins, OMVs make for an excellent native-like

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    Cleavable Affinity Purification (Cl-AP): A One-step Procedure to Affinity Purify Protein Complexes
    Authors:  Ammarah Tariq, Lucy Green, Christian Soeller and James G. Wakefield, date: 11/20/2020, view: 1738, Q&A: 0
    [Abstract]

    Cleavable Affinity Purification (Cl-AP) uses a tripartite system of Protein-A-Streptavidin beads and nanobodies, coupled with a biotinylated, thiol-cleavable linker, providing one-step affinity purification from lysates of tissues expressing tagged proteins. This technique allows fluorescent versions of mitotic protein complexes to be isolated

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    Expression and Purification of Functionally Active Serotonin 5-HT2A Receptor in Insect Cells Using Low-titer Viral Stock
    [Abstract] The serotonin 5-HT2A receptor (5-HT2AR) is a member of the GPCR family that is important for various neurological functions and whose dysregulation causes many mental health disorders. Structural investigations of 5-HT2AR require the production of functionally active receptors expressed from eukaryotic cell ...
    Production and Isolation of Magnetic Protein Crystals in HEK293T Cells
    Authors:  Thomas L. Li and Bianxiao Cui, date: 07/20/2020, view: 1747, Q&A: 0
    [Abstract] Advances in protein engineering have enabled the production of self-assembled protein crystals within living cells. Our recent publication demonstrates the production of ftn-PAK4, which is a ferritin-containing crystal that can mineralize iron and become magnetic when isolated. We have developed an optimized protocol for the production and ...
    Mouse Adipose Tissue Protein Extraction
    Authors:  Yu A. An and Philipp E. Scherer, date: 06/05/2020, view: 4510, Q&A: 0
    [Abstract] As obesity becomes a global epidemic, the metabolism research field is increasingly focusing on studying the physiological and pathological roles of adipose tissues (AT). However, extracting proteins from AT is challenging due to abundant fat content of intracellular lipid droplets. Several commercial kits for extraction of AT proteins are ...



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