Biochemistry

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    Protocols in Current Issue
    Self-organization Assay for Min Proteins of Escherichia coli in Micro-droplets Covered with Lipids
    Authors:  Shunshi Kohyama, Kei Fujiwara, Natsuhiko Yoshinaga and Nobuhide Doi, date: 03/20/2020, view: 416, Q&A: 0
    [Abstract] The Min system determines the cell division plane of bacteria. As a cue of spatiotemporal regulation, the Min system uses wave propagation of MinD protein (Min wave). Therefore, the reconstitution of the Min wave in cell-sized closed space will lead to the creation of artificial cells capable of cell division. The Min waves emerge via coupling ...
    Production, Purification and Characterization of Recombinant Biotinylated Phytochrome B for Extracellular Optogenetics
    Authors:  Maximilian Hörner, O. Sascha Yousefi, Wolfgang W. A. Schamel and Wilfried Weber, date: 03/05/2020, view: 608, Q&A: 0
    [Abstract] In the field of extracellular optogenetics, photoreceptors are applied outside of cells to obtain systems with a desired functionality. Among the diverse applied photoreceptors, phytochromes are the only ones that can be actively and reversibly switched between the active and inactive photostate by the illumination with cell-compatible red and ...
    Quantification of Protein Enrichment at Plasmodesmata
    Authors:  Magali S. Grison, Jules D. Petit, Marie Glavier and Emmanuelle M. Bayer, date: 03/05/2020, view: 560, Q&A: 0
    [Abstract] Intercellular communication plays a crucial role in the establishment of multicellular organisms by organizing and coordinating growth, development and defence responses. In plants, cell-to-cell communication takes place through nanometric membrane channels called plasmodesmata (PD). Understanding how PD dictate cellular connectivity greatly ...
    Expression and Purification of Adeno-associated Virus Virus-like Particles in a Baculovirus System and AAVR Ectodomain Constructs in E. coli
    [Abstract] Adeno-associated virus (AAV) is a promising gene therapy vector and the biophysical characterization of its interactions with host proteins is a critical foundation for engineering tissue targeting and immune escape. Presented here are protocols for the production of: (a) the outer protein shells (virus-like particles or VLPs) for serotype 2 ...
    Preparation and Purification of Active Recombinant Human Pancreatic Lipase in Escherichia coli
    Authors:  Nanami Kawaguchi, Haruko Ogawa and Kimie Date, date: 07/05/2019, view: 1358, Q&A: 0
    [Abstract] Human pancreatic lipase (HPL) is the main lipolytic enzyme involved in the digestion of dietary fat. An active recombinant human pancreatic lipase (recHPL) was successfully prepared for the first time in an Escherichia coli (E. coli) expression system using a short Strep-tag II (ST II). The recHPL-ST II was solubilized with 8 M urea from ...
    A Detailed Protocol for Large-scale Recombinant Expression and Validation of Human FGFR2 with Its Transmembrane and Extracellular Domains in Escherichia coli
    Authors:  Adam Bajinting and Ho Leung Ng, date: 06/05/2019, view: 1795, Q&A: 0
    [Abstract] Receptor tyrosine kinases (RTKs) are an important class of transmembrane receptors that mediate some of the most crucial biochemical pathways essential to the growth, differentiation, and survival of a cell and thus, are highly involved in cancers. Due to the complexity of RTKs having biochemically different domains including a transmembrane ...
    Expression and Ni-NTA-Agarose Purification of Recombinant Hepatitis C Virus E2 Ectodomain Produced in a Baculovirus Expression System
    [Abstract] In this protocol, we describe the production and purification of the ectodomain of the E2661 envelope protein (amino acids 384-661) of the Hepatitis C virus, which plays a fundamental role in the entry of the virus into the host cell. This protein has been expressed in both prokaryotic and eukaryotic systems but in small quantities or ...
    Detection of Membrane Protein Interactions by Cell-based Tango Assays
    [Abstract] The Tango assay is a protein-protein interaction assay, in which a transcription factor (rTA) is fused to a membrane-bound protein via a linker that contains a cleavage site for TEV protease, whereas a soluble interaction partner is fused to TEV protease (Barnea et al., 2008). Association between the two interaction partners leads to an ...
    Protein Expression and Purification of the Hsp90-Cdc37-Cdk4 Kinase Complex from Saccharomyces cerevisiae
    Authors:  Kliment A. Verba and David A. Agard, date: 10/05/2017, view: 5111, Q&A: 0
    [Abstract] Interactions between Hsp90, its co-chaperone Cdc37 and kinases have been biochemically studied for over three decades and have been shown to be functionally important in organisms from yeast to humans. However, formation of a stable complex for structural studies has been elusive. In this protocol we describe expression and purification of ...
    Expression and Purification of a Mammalian P2X7 Receptor from Sf9 Insect Cells
    Authors:  Akira Karasawa and Toshimitsu Kawate, date: 09/05/2017, view: 6598, Q&A: 0
    [Abstract] The P2X7 receptor is an extracellular ATP-gated ion channel found only in eukaryotes (Bartlett et al., 2014). Due to its unique properties among P2X receptors, such as formation of a large conductance pore, the P2X7 receptor has been implicated in devastating diseases like chronic pain (North and Jarvis, 2013). However, mechanisms ...



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