Prokaryotes

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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: 418, Q&A: 0
    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 ...
    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: 506, Q&A: 0
    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 ...
    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: 418, 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: 506, 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 ...
    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: 365, 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 ...
    HoSeIn: A Workflow for Integrating Various Homology Search Results from Metagenomic and Metatranscriptomic Sequence Datasets
    Authors:  Gaston Rozadilla, Jorgelina Moreiras Clemente and Christina B. McCarthy, date: 07/20/2020, view: 311, Q&A: 0
    [Abstract] Data generated by metagenomic and metatranscriptomic experiments is both enormous and inherently noisy. When using taxonomy-dependent alignment-based methods to classify and label reads, the first step consists in performing homology searches against sequence databases. To obtain the most information from the samples, nucleotide sequences are ...
    Preparation of Yeast tRNA Sample for NMR Spectroscopy
    Authors:  Marjorie Catala, Alexandre Gato, Carine Tisné and Pierre Barraud, date: 06/20/2020, view: 647, Q&A: 0
    [Abstract] Transfer RNAs (tRNAs) are heavily decorated with post-transcriptional modifications during their biosynthesis. To fulfil their functions within cells, tRNAs undergo a tightly controlled biogenesis process leading to the formation of mature tRNAs. In addition, functions of tRNAs are often modulated by their modifications. Although the biological ...
    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: 829, 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: 897, 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 ...
    Flow Cytometry Analysis and Fluorescence-activated Cell Sorting of Myeloid Cells from Lung and Bronchoalveolar Lavage Samples from Mycobacterium tuberculosis-infected Mice
    Authors:  Alissa C. Rothchild, Dat Mai, Alan Aderem and Alan H. Diercks, date: 05/20/2020, view: 1194, Q&A: 0
    [Abstract] Mycobacterium tuberculosis (Mtb) is transmitted by aerosol and can cause serious bacterial infection in the lung that can be fatal if left untreated. Mtb is now the leading cause of death worldwide by an infectious agent. Characterizing the early events of in vivo infection following aerosol challenge is critical for ...
    Purification of Protein-complexes from the Cyanobacterium Synechocystis sp. PCC 6803 Using FLAG-affinity Chromatography
    Authors:  Minna M. Koskela, Petra Skotnicová, Éva Kiss and Roman Sobotka, date: 05/20/2020, view: 962, Q&A: 0
    [Abstract] Exploring the structure and function of protein complexes requires their isolation in the native state–a task that is made challenging when studying labile and/or low abundant complexes. The difficulties in preparing membrane-protein complexes are especially notorious. The cyanobacterium Synechocystis sp. PCC 6803 is a widely used model ...
    Molecular Size Analysis of Recombinant Importin-histone Complexes Using Analytical Ultracentrifugation
    Authors:  Abhilash Padavannil, Chad A. Brautigam and Yuh Min Chook, date: 05/20/2020, view: 838, Q&A: 0
    [Abstract] Histones constitute the protein components of nucleosomes. Despite their small sizes, histones do not diffuse through the nuclear pore complex. Instead, they are transported to the nucleus by importins, either alone or in complex with histone chaperones. Determining the molecular size of the importin-histone complexes is key to understanding the ...



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