Biochemistry

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    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: 714, 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: 794, 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: 4291, 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: 5496, 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 ...
    Expression and Purification of Mini G Proteins from Escherichia coli
    Authors:  Byron Carpenter and Christopher G. Tate, date: 04/20/2017, view: 6296, Q&A: 0
    [Abstract] Heterotrimeric G proteins modulate intracellular signalling by transducing information from cell surface G protein-coupled receptors (GPCRs) to cytoplasmic effector proteins. Structural and functional characterisation of GPCR–G protein complexes is important to fully decipher the mechanism of signal transduction. However, native G proteins are ...
    Production, Purification and Crystallization of a Prokaryotic SLC26 Homolog for Structural Studies
    Authors:  Yung-Ning Chang, Farooque R. Shaik, Yvonne Neldner and Eric R. Geertsma, date: 02/05/2017, view: 4666, Q&A: 0
    [Abstract] The SLC26 or SulP proteins constitute a large family of anion transporters that are ubiquitously expressed in pro- and eukaryotes. In human, SLC26 proteins perform important roles in ion homeostasis and malfunctioning of selected members is associated with diseases. This protocol details the production and crystallization of a prokaryotic SLC26 ...
    Expression and Purification of the GRAS Domain of Os-SCL7 from Rice for Structural Studies
    Authors:  Shengping Li, Yanhe Zhao and Yunkun Wu, date: 02/05/2017, view: 4226, Q&A: 0
    [Abstract] GRAS proteins, named after the first three members GAI, RGA and SRC, has been found in 294 embryophyta species and is represented by 1,035 sequences. They belong to a plant-specific protein family and play essential roles in plant growth and development. Proteins in this family are defined as minimally containing a conserved GRAS domain, which is ...
    Expression, Purification and Crystallization of Recombinant Arabidopsis Monogalactosyldiacylglycerol Synthase (MGD1)
    Authors:  Joana Rocha, Valerie Chazalet and Christelle Breton, date: 12/20/2016, view: 4250, Q&A: 0
    [Abstract] In plant cells, galactolipids are predominant, representing up to 50% of the lipid content in photosynthetic tissues. Galactolipid synthesis is initiated by MGDG synthases (MGDs), which use UDP-galactose as a donor sugar and diacylglycerol (DAG) as acceptor, to form monogalactosyldiacylglycerol (MGDG). This protocol is used to produce a ...



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