Molecular Biology

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    Protocols in Current Issue
    High-level Production of Recombinant Membrane Proteins Using the Engineered Escherichia coli Strains SuptoxD and SuptoxR
    Authors:  Myrsini Michou, Dafni C. Delivoria and Georgios Skretas, date: 08/05/2020, view: 404, Q&A: 0
    [Abstract] We have previously described the development of two specialized Escherichia coli strains for high-level recombinant membrane protein (MP) production. These engineered strains, termed SuptoxD and SuptoxR, are capable of suppressing the cytotoxicity caused by MP overexpression and of producing greatly enhanced MP yields. Here, we present a ...
    A Method for User-defined Mutagenesis by Integrating Oligo Pool Synthesis Technology with Nicking Mutagenesis
    Authors:  Paul J. Steiner, Zachary T Baumer and Timothy A. Whitehead, date: 08/05/2020, view: 477, Q&A: 0
    [Abstract] Saturation mutagenesis is a fundamental enabling technology for protein engineering and epitope mapping. Nicking mutagenesis (NM) allows the user to rapidly construct libraries of all possible single mutations in a target protein sequence from plasmid DNA in a one-pot procedure. Briefly, one strand of the plasmid DNA is degraded using a nicking ...
    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 ...
    A SsrA/NIa-based Strategy for Post-Translational Regulation of Protein Levels in Gram-negative Bacteria
    Authors:  Gonzalo Durante-Rodríguez, Belén Calles, Víctor de Lorenzo and Pablo I. Nikel, date: 07/20/2020, view: 362, Q&A: 0
    [Abstract] Strategies to control the levels of key enzymes of bacterial metabolism are commonly based on the manipulation of gene of interest within the target pathway. The development of new protocols towards the manipulation of biochemical processes is still a major challenge in the field of metabolic engineering. On this background, the FENIX (functional ...
    Generation and Testing of Fluorescent Adaptable Simple Theranostic (FAST) Proteins
    [Abstract] This protocol provides a step-by-step method to create recombinant fluorescent fusion proteins that can be secreted from mammalian cell lines. This builds on many other recombinant protein and fluorescent protein techniques, but is among the first to harness fluorescent fusion proteins secreted directly into cell culture supernatant. This opens ...
    In vitro Assessment of Pathogen Effector Binding to Host Proteins by Surface Plasmon Resonance
    [Abstract] The mechanisms of virulence and immunity are often governed by molecular interactions between pathogens and host proteins. The study of these interactions has major implications on understanding virulence activities, and how the host immune system recognizes the presence of pathogens to initiate an immune response. Frequently, the association ...
    Superresolution Microscopy of Drosophila Indirect Flight Muscle Sarcomeres
    Authors:  Szilárd Szikora, Tibor Novák, Tamás Gajdos, Miklós Erdélyi and József Mihály, date: 06/20/2020, view: 669, Q&A: 0
    [Abstract] Sarcomeres are extremely highly ordered macromolecular assemblies where proper structural organization is an absolute prerequisite to the functionality of these contractile units. Despite the wealth of information collected, the exact spatial arrangement of many of the H-zone and Z-disk proteins remained unknown. Recently, we developed a powerful ...
    Site-specific DNA Mapping of Protein Binding Orientation Using Azidophenacyl Bromide (APB)
    Authors:  Himasha M. Perera and Michael A. Trakselis, date: 06/20/2020, view: 824, Q&A: 0
    [Abstract] The orientation of a DNA-binding protein bound on DNA is determinative in directing the assembly of other associated proteins in the complex for enzymatic action. As an example, in a replisome, the orientation of the DNA helicase at the replication fork directs the assembly of the other associated replisome proteins. We have recently determined ...
    Electrophoretic Mobility Shift Assay of in vitro Phosphorylated RNA Polymerase II Carboxyl-terminal Domain Substrates
    Authors:  Joshua E. Mayfield, Seema Irani and Yan Zhang, date: 06/20/2020, view: 886, Q&A: 0
    [Abstract] Eukaryotic RNA polymerase II transcribes all protein-coding mRNAs and is highly regulated. A key mechanism directing RNA polymerase II and facilitating the co-transcriptional processing of mRNAs is the phosphorylation of its highly repetitive carboxyl-terminal domain (CTD) of its largest subunit, RPB1, at specific residues. A variety of techniques ...
    Quantitative Nucleocytoplasmic Transport Assays in Cellular Models of Neurodegeneration
    [Abstract] Nucleocytoplasmic transport deficits are suggested to play a role in neurodegenerative disorders, including amyotrophic lateral sclerosis (ALS). Given the importance and complexity of this process, understanding when these aberrations occur and which pathways are involved is of great importance. Here, we make use of CRISPR-Cas9 technology to ...



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