DOCSLIB.ORG

Development of an Enzyme-Based Method for Production of Food with Low Purine Content

Total Page：16

File Type：pdf, Size：1020Kb

Download full-text PDF Read full-text
Development of an Enzyme-Based Method for Production of Food with Low Purine Content Development of an enzyme-based method for production of food with low purine content I n a u g u r a l d i s s e r t a t i o n zur Erlangung des akademischen Grades eines Doktors der Naturwissenschaften (Dr. rer. nat.) der Mathematisch-Naturwissenschaftlichen Fakultät der Ernst-Moritz-Arndt-Universität Greifswald vorgelegt von Dagmara Jankowska geboren am 27. Juni 1984 in Wrocław, Polen - Greifswald, 2013 - Dekan: Prof. Dr. Klaus Fesser 1. Gutachter : Prof. Dr. habil. Rϋdiger Bode Universität Greifswald, Institut für Biochemie 2. Gutachter: Prof. Dr. Raffael Schaffrath Universität Kassel, Institut für Biologie Tag der Promotion: 14.04.2014 Table of contents TABLE OF CONTENTS Summary ............................................................................................................................ i Zusammenfassung ............................................................................................................ iii List of abbreviations .......................................................................................................... v 1 Introduction .................................................................................................................. 1 1.1 Purine degradation pathway ........................................................................................ 1 1.1.1 Purines .............................................................................................................. 1 1.1.2 Purine degradation ........................................................................................... 2 1.2 Why do humans lack urate oxidase? ........................................................................... 4 1.3 Purines in food products .............................................................................................. 5 1.4 Uric acid and hyperuricemia ........................................................................................ 5 1.5 Gout .............................................................................................................................. 7 1.6 Treatment of hyperuricemia and gout ......................................................................... 9 1.6.1 Acute gout treatment ..................................................................................... 10 1.6.2 Uricostatic drugs ............................................................................................. 12 1.6.3 Uricosuric drugs .............................................................................................. 12 1.6.4 Uricolytic drugs ............................................................................................... 13 1.6.5 Prevention of gout .......................................................................................... 13 1.7 Adenine deaminase .................................................................................................... 14 1.8 Xanthine oxidoreductase ........................................................................................... 15 1.9 Arxula adeninivorans .................................................................................................. 16 1.10 Xplor®2 transformation/expression platform ............................................................ 17 2 Aim of the thesis ......................................................................................................... 20 3 Materials and methods ............................................................................................... 21 3.1 Microorganisms .......................................................................................................... 21 3.2 Oligonucleotides ......................................................................................................... 21 3.3 Plasmids and vectors .................................................................................................. 22 3.4 Culture media ............................................................................................................. 22 3.4.1 Bacterial media ............................................................................................... 22 3.4.2 Yeast media .................................................................................................... 23 3.5 Enzymes and chemicals .............................................................................................. 24 3.6 Cultivation of microorganisms ................................................................................... 24 3.6.1 Cultivation of E. coli cells ................................................................................ 24 3.6.2 Cultivation of yeast cells ................................................................................. 25 3.7 Methods used for work with DNA .............................................................................. 25 3.7.1 Agarose gel electrophoresis ........................................................................... 25 3.7.2 DNA fragment extraction from agarose gel ................................................... 25 Table of contents 3.7.3 Mini isolation of plasmid DNA from E. coli cells ............................................ 26 3.7.4 DNA restriction ............................................................................................... 26 3.7.5 DNA ligation ................................................................................................... 26 3.7.6 Ethanol precipitation of DNA ......................................................................... 26 3.7.7 Transformation of E. coli cells ........................................................................ 27 3.7.8 Preparation of E. coli competent cells ........................................................... 27 3.7.9 Polymerase Chain Reaction (PCR) .................................................................. 27 3.7.10 Southern blotting ........................................................................................... 28 3.8 Methods used for work with RNA ............................................................................. 29 3.8.1 RNA isolation from yeast cells ....................................................................... 29 3.8.2 Electrophoresis of RNA .................................................................................. 30 3.8.3 Removal of genomic DNA from RNA samples ............................................... 30 3.8.4 cDNA synthesis ............................................................................................... 31 3.8.5 Real-time quantitative PCR ............................................................................ 31 3.9 Methods used for work with proteins ....................................................................... 31 3.9.1 Determination of protein concentration ....................................................... 31 3.9.2 Protein electrophoresis in a polyacrylamide gel (SDS-PAGE) ........................ 32 3.9.3 Protein electrophoresis in a native polyacrylamide gel................................. 33 3.9.4 Western blotting (immunoblotting) .............................................................. 33 3.10 Protein purification .................................................................................................... 35 3.10.1 Cell wall disruption ......................................................................................... 35 3.10.2 Affinity chromatography (Ni-NTA chromatography) ..................................... 35 3.10.3 Ion exchange and gel filtration ...................................................................... 36 3.11 N-terminal sequencing............................................................................................... 36 3.12 MALDI-TOF ................................................................................................................. 37 3.13 Methods used for work with yeast ............................................................................ 37 3.13.1 Transformation of yeast cells ......................................................................... 37 3.13.2 Preparation of competent A. adeninivorans G1212 cells .............................. 38 3.13.3 Stabilization of insert in the yeast genomic DNA .......................................... 38 3.13.4 Isolation of yeast chromosomal DNA ............................................................ 39 3.14 Expression of wild-type proteins, Aadap and Axorp, in A. adeninivorans LS3 .......... 39 3.15 Cultivation of transgenic strains for protein expression ........................................... 40 3.15.1 E. coli .............................................................................................................. 40 3.15.2 A. adeninivorans ............................................................................................. 40 3.16 Subcellular fractionation ............................................................................................ 40 3.17 Yeast cells microscopy ............................................................................................... 41 3.18 Cell growth monitoring .............................................................................................. 41 3.18.1 Optical density of the culture ........................................................................ 41 3.18.2 Dry cell weight ................................................................................................ 41 3.19 Enzymatic assays .......................................................................................................
Load more

Recommended publications
  • Structural Insights Into Histone Modifying Enzymes
    Wayne State University Wayne State University Theses January 2019 Structural Insights Into Histone Modifying Enzymes Shruti Amle Wayne State University, [email protected] Follow this and additional works at: https://digitalcommons.wayne.edu/oa_theses Part of the Biochemistry Commons, and the Molecular Biology Commons Recommended Citation Amle, Shruti, "Structural Insights Into Histone Modifying Enzymes" (2019). Wayne State University Theses. 693. https://digitalcommons.wayne.edu/oa_theses/693 This Open Access Embargo is brought to you for free and open access by DigitalCommons@WayneState. It has been accepted for inclusion in Wayne State University Theses by an authorized administrator of DigitalCommons@WayneState. STRUCTURAL INSIGHTS INTO HISTONE MODIFYING ENZYMES by SHRUTI AMLE THESIS Submitted to the Graduate School of Wayne State University, Detroit, Michigan in partial fulfillment of the requirements for the degree of MASTER OF SCIENCE 2019 MAJOR: BIOCHEMISTRY AND MOLECULAR BIOLOGY Approved By: _________________________________________ Advisor Date _________________________________________ _________________________________________ _________________________________________ i ACKNOWLEDGEMENTS Writing this thesis has been extremely captivating and gratifying. I take this opportunity to express my deep and sincere acknowledgements to the number of people for extending their generous support and unstinted help during my entire study. Firstly, I would like to express my respectful regards and deep sense of gratitude to my advisor, Dr. Zhe Yang. I am extremely honored to study and work under his guidance. His vision, ideals, timely motivation and immense knowledge had a deep influence on my entire journey of this career. Without his understanding and support, it would not have been possible to complete this research successfully. I also owe my special thanks to my committee members: Dr.
    [Show full text]
  • Arxula Adeninivorans
    Biernacki et al. Microb Cell Fact (2017) 16:144 DOI 10.1186/s12934-017-0751-4 Microbial Cell Factories RESEARCH Open Access Enhancement of poly(3‑hydroxybutyrate‑co‑ 3‑hydroxyvalerate) accumulation in Arxula adeninivorans by stabilization of production Mateusz Biernacki1, Marek Marzec1,6, Thomas Roick2, Reinhard Pätz3, Kim Baronian4, Rüdiger Bode5 and Gotthard Kunze1* Abstract Background: In recent years the production of biobased biodegradable plastics has been of interest of research- ers partly due to the accumulation of non-biodegradable plastics in the environment and to the opportunity for new applications. Commonly investigated are the polyhydroxyalkanoates (PHAs) poly(hydroxybutyrate) and poly(hydroxybutyrate-co-hydroxyvalerate) (PHB-V). The latter has the advantage of being tougher and less brittle. The production of these polymers in bacteria is well established but production in yeast may have some advantages, e.g. the ability to use a broad spectrum of industrial by-products as a carbon sources. Results: In this study we increased the synthesis of PHB-V in the non-conventional yeast Arxula adeninivorans by stabilization of polymer accumulation via genetic modifcation and optimization of culture conditions. An A. adenini- vorans strain with overexpressed PHA pathway genes for β-ketothiolase, acetoacetyl-CoA reductase, PHAs synthase and the phasin gene was able to accumulate an unexpectedly high level of polymer. It was found that an opti- 1 mized strain cultivated in a shaking incubator is able to produce up to 52.1% of the DCW of PHB-V (10.8 g L− ) with 12.3%mol of PHV fraction. Although further optimization of cultivation conditions in a fed-batch bioreactor led to lower polymer content (15.3% of the DCW of PHB-V), the PHV fraction and total polymer level increased to 23.1%mol 1 and 11.6 g L− respectively.
    [Show full text]
  • Structural Properties of the Nickel Ions in Urease: Novel Insights Into the Catalytic and Inhibition Mechanisms
    Coordination Chemistry Reviews 190–192 (1999) 331–355 www.elsevier.com/locate/ccr Structural properties of the nickel ions in urease: novel insights into the catalytic and inhibition mechanisms Stefano Ciurli a,*, Stefano Benini b, Wojciech R. Rypniewski b, Keith S. Wilson c, Silvia Miletti a, Stefano Mangani d a Institute of Agricultural Chemistry, Uni6ersity of Bologna, Viale Berti Pichat 10, I-40127 Bologna, Italy b European Molecular Biology Laboratory, c/o DESY, Notkestraße 85, D-22603 Hamburg, Germany c Department of Chemistry, Uni6ersity of York, Heslington, York YO15DD, UK d Department of Chemistry, Uni6ersity of Siena, Pian dei Mantellini 44, I-53100 Siena, Italy Accepted 13 March 1999 Contents Abstract.................................................... 331 1. Biological background ......................................... 332 2. Spectroscopic investigations of the urease active site structure .................. 333 3. Crystallographic studies of the native enzyme ............................ 334 4. Crystallographic studies of urease mutants.............................. 341 5. Crystallographic studies of urease–inhibitor complexes ...................... 345 6. Crystallographic study of a transition state analogue bound to urease.............. 348 7. A novel proposal for the urease mechanism ............................. 350 References .................................................. 353 Abstract This work provides a comprehensive critical summary of urease spectroscopy, crystallogra- phy, inhibitor binding, and site-directed
    [Show full text]
  • The Microbiota-Produced N-Formyl Peptide Fmlf Promotes Obesity-Induced Glucose
    Page 1 of 230 Diabetes Title: The microbiota-produced N-formyl peptide fMLF promotes obesity-induced glucose intolerance Joshua Wollam1, Matthew Riopel1, Yong-Jiang Xu1,2, Andrew M. F. Johnson1, Jachelle M. Ofrecio1, Wei Ying1, Dalila El Ouarrat1, Luisa S. Chan3, Andrew W. Han3, Nadir A. Mahmood3, Caitlin N. Ryan3, Yun Sok Lee1, Jeramie D. Watrous1,2, Mahendra D. Chordia4, Dongfeng Pan4, Mohit Jain1,2, Jerrold M. Olefsky1 * Affiliations: 1 Division of Endocrinology & Metabolism, Department of Medicine, University of California, San Diego, La Jolla, California, USA. 2 Department of Pharmacology, University of California, San Diego, La Jolla, California, USA. 3 Second Genome, Inc., South San Francisco, California, USA. 4 Department of Radiology and Medical Imaging, University of Virginia, Charlottesville, VA, USA. * Correspondence to: 858-534-2230, [email protected] Word Count: 4749 Figures: 6 Supplemental Figures: 11 Supplemental Tables: 5 1 Diabetes Publish Ahead of Print, published online April 22, 2019 Diabetes Page 2 of 230 ABSTRACT The composition of the gastrointestinal (GI) microbiota and associated metabolites changes dramatically with diet and the development of obesity. Although many correlations have been described, specific mechanistic links between these changes and glucose homeostasis remain to be defined. Here we show that blood and intestinal levels of the microbiota-produced N-formyl peptide, formyl-methionyl-leucyl-phenylalanine (fMLF), are elevated in high fat diet (HFD)- induced obese mice. Genetic or pharmacological inhibition of the N-formyl peptide receptor Fpr1 leads to increased insulin levels and improved glucose tolerance, dependent upon glucagon- like peptide-1 (GLP-1). Obese Fpr1-knockout (Fpr1-KO) mice also display an altered microbiome, exemplifying the dynamic relationship between host metabolism and microbiota.
    [Show full text]
  • Supplementary Information
    Supplementary information (a) (b) Figure S1. Resistant (a) and sensitive (b) gene scores plotted against subsystems involved in cell regulation. The small circles represent the individual hits and the large circles represent the mean of each subsystem. Each individual score signifies the mean of 12 trials – three biological and four technical. The p-value was calculated as a two-tailed t-test and significance was determined using the Benjamini-Hochberg procedure; false discovery rate was selected to be 0.1. Plots constructed using Pathway Tools, Omics Dashboard. Figure S2. Connectivity map displaying the predicted functional associations between the silver-resistant gene hits; disconnected gene hits not shown. The thicknesses of the lines indicate the degree of confidence prediction for the given interaction, based on fusion, co-occurrence, experimental and co-expression data. Figure produced using STRING (version 10.5) and a medium confidence score (approximate probability) of 0.4. Figure S3. Connectivity map displaying the predicted functional associations between the silver-sensitive gene hits; disconnected gene hits not shown. The thicknesses of the lines indicate the degree of confidence prediction for the given interaction, based on fusion, co-occurrence, experimental and co-expression data. Figure produced using STRING (version 10.5) and a medium confidence score (approximate probability) of 0.4. Figure S4. Metabolic overview of the pathways in Escherichia coli. The pathways involved in silver-resistance are coloured according to respective normalized score. Each individual score represents the mean of 12 trials – three biological and four technical. Amino acid – upward pointing triangle, carbohydrate – square, proteins – diamond, purines – vertical ellipse, cofactor – downward pointing triangle, tRNA – tee, and other – circle.
    [Show full text]
  • Yeasts of the Blastobotrys Genus Are Promising Platform for Lipid-Based
    Yeasts of the Blastobotrys genus are promising platform for lipid-based fuels and oleochemicals production Daniel Sanya, Djamila Onésime, Volkmar Passoth, Mrinal Maiti, Atrayee Chattopadhyay, Mahesh Khot To cite this version: Daniel Sanya, Djamila Onésime, Volkmar Passoth, Mrinal Maiti, Atrayee Chattopadhyay, et al.. Yeasts of the Blastobotrys genus are promising platform for lipid-based fuels and oleochemicals production. Applied Microbiology and Biotechnology, Springer Verlag, 2021, 105, pp.4879 - 4897. 10.1007/s00253-021-11354-3. hal-03274326 HAL Id: hal-03274326 https://hal.inrae.fr/hal-03274326 Submitted on 30 Jun 2021 HAL is a multi-disciplinary open access L’archive ouverte pluridisciplinaire HAL, est archive for the deposit and dissemination of sci- destinée au dépôt et à la diffusion de documents entific research documents, whether they are pub- scientifiques de niveau recherche, publiés ou non, lished or not. The documents may come from émanant des établissements d’enseignement et de teaching and research institutions in France or recherche français ou étrangers, des laboratoires abroad, or from public or private research centers. publics ou privés. Applied Microbiology and Biotechnology https://doi.org/10.1007/s00253-021-11354-3 MINI-REVIEW Yeasts of the Blastobotrys genus are promising platform for lipid-based fuels and oleochemicals production Daniel Ruben Akiola Sanya1 & Djamila Onésime1 & Volkmar Passoth2 & Mrinal K. Maiti3 & Atrayee Chattopadhyay3 & Mahesh B. Khot4 Received: 18 February 2021 /Revised: 29 April 2021 /Accepted: 16 May 2021 # The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature 2021 Abstract Strains of the yeast genus Blastobotrys (subphylum Saccharomycotina) represent a valuable biotechnological resource for basic biochemistry research, single-cell protein, and heterologous protein production processes.
    [Show full text]
  • Investigation of Agonistic and Antagonistic Endocrine Activity During Full-Scale Ozonation of Waste Water
    Investigation of agonistic and antagonistic endocrine activity during full-scale ozonation of waste water Dissertation zur Erlangung des akademischen Grades eines Doktors der Naturwissenschaften – Dr. rer. nat. – vorgelegt von Fabian Itzel geboren in Moers Fakultät für Chemie der Universität Duisburg-Essen 2018 Die vorliegende Arbeit wurde im Zeitraum von September 2014 bis September 2018 im Arbeitskreis von Prof. Dr. Torsten C. Schmidt in der Fakultät für Chemie im Bereich Instrumentelle Analytische Chemie der Universität Duisburg-Essen durchgeführt. Tag der Disputation: 21.02.2019 Gutachter: Prof. Dr. Torsten C. Schmidt Prof. Dr. Elke Dopp Vorsitzender: Prof. Dr. Bettina Siebers I Summary The use of a wide variety of chemicals in our society, such as industrial chemicals, pharmaceuticals, personal care products, etc., leads to pollution of surface waters. Especially in densely populated urban areas such as the Ruhr catchment, sustainable water management poses a major challenge. Despite intensive use through various types of discharges (effluents of direct dischargers, municipal waste water treatment plants, industry, etc.), good water quality has always to be guaranteed in accordance to the European Water Framewor Directive. Endocrine disrupting chemicals can have an effect on aquatic organisms even at very low concentrations (pg/L range). In order to reduce the emission, ozonation was investigated as advanced waste water treatment for the elimination of organic trace compounds. An elimination performance of ≥ 80% for selected substances at specific ozone doses in the range of zspec. = 0.3 - 0.7 mgO3/mgDOC was achieved. Since 2015, estrogens are listed on the watch-list of the European Water Framework Directive with required detection limits in the pg/L range.
    [Show full text]
  • PURINE SALVAGE in HELICOBACTER PYLORI by ERICA FRANCESCA MILLER (Under the Direction of Robert J. Maier) ABSTRACT Purines Are Es
    PURINE SALVAGE IN HELICOBACTER PYLORI by ERICA FRANCESCA MILLER (Under the Direction of Robert J. Maier) ABSTRACT Purines are essential for all living cells. This fact is reflected in the high degree of pathway conservation for purine metabolism across all domains of life. The availability of purines within a mammalian host is thought to be a limiting factor for infection, as demonstrated by the importance of purine synthesis and salvage genes among many bacterial pathogens. Helicobacter pylori, a primary causative agent of peptic ulcers and gastric cancers, colonizes a niche that is otherwise uninhabited by bacteria: the surface of the human gastric epithelium. Despite many studies over the past 30 years that have addressed virulence mechanisms such as acid resistance, little knowledge exists regarding this organism’s purine metabolism. To fill this gap in knowledge, we asked whether H. pylori can carry out de novo purine biosynthesis, and whether its purine salvage network is complete. Based on genomic data from the fully sequenced H. pylori genomes, we combined mutant analysis with physiological studies to determine that H. pylori, by necessity, must acquire purines from its human host. Furthermore, we found the purine salvage network to be complete, allowing this organism to use any single purine nucleobase or nucleoside for growth. In the process of elucidating these pathways, we discovered a nucleoside transporter in H. pylori that, in contrast to the biochemically- characterized homolog NupC, aids in uptake of purine rather than pyrimidine nucleosides into the cell. Lastly, we investigated an apparent pathway gap in the genome annotation—that of adenine degradation—and in doing so uncovered a new family of adenosine deaminase that lacks sequence homology with all other adenosine deaminases studied to date.
    [Show full text]
  • Optimización De La Producción De Riboflavina En Ashbya Gossypii Basada En El Análisis Funcional De Las Rutas De Recuperación De Nucleobases De Purina
    Universidad de Salamanca Departamento de Microbiología y Genética Optimización de la producción de riboflavina en Ashbya gossypii basada en el análisis funcional de las rutas de recuperación de nucleobases de purina. José Antonio Uña Álvarez Tesis Doctoral 2013 Dr. Luís Román Fernández Lago, Director del Departamento de Microbiología y Genética de la Universidad de Salamanca. CERTIFICA: Que la memoria titulada “Optimización de la producción de riboflavina en Ashbya gossypii basada en el análisis funcional de las rutas de recuperación de nucleobases de purina”, presentada por D. José Antonio Uña Álvarez para optar al grado de Doctor en Biología, ha sido realizada bajo la dirección de la Dra. Mª Ángeles Santos García y del Dr. José Luis Revuelta Doval, en el Departamento de Microbiología y Genética de la Universidad de Salamanca. Y para que así conste expide el presenta certificado en: Salamanca a de de 2013 Fdo. Dr. Luís Román Fernández Lago Dra. Mª Ángeles Santos García, Profesora Titular del Departamento de Microbiología y Genética de la Universidad de Salamanca y Dr. José Luis Revuelta Doval, Catedrático de Genética del Departamento de Microbiología y Genética de la Universidad de Salamanca. CERTIFICAN: Que el doctorando José Antonio Uña Álvarez ha realizado en el Departamento de Microbiología y Genética de la Universidad de Salamanca, bajo su dirección, el trabajo titulado “Optimización de la producción de riboflavina en Ashbya gossypii basada en el análisis funcional de las rutas de recuperación de nucleobases de purina”, para optar al grado de Doctor en Biología. Y para que así conste, y como directores del trabajo, expiden el presente certificado en: Salamanca a de de 2013 Fdo: Dra.
    [Show full text]
  • Purine and Pyrimidine Metabolism in Human Epidermis* Jean De Bersaques, Md
    THE JOURNAL OP INVESTIGATIVE DERMATOLOGY Vol. 4s, No. Z Copyright 1957 by The Williams & Wilkins Co. Fri nte,1 in U.S.A. PURINE AND PYRIMIDINE METABOLISM IN HUMAN EPIDERMIS* JEAN DE BERSAQUES, MD. The continuous cellular renewal occurring inthine, which contained 5% impurity, and for uric the epidermis requires a very active synthesisacid, which consisted of 3 main components. The reaction was stopped after 1—2 hours in- and breakdown of nuclear and cytoplasmiecubation at 37° and the products were spotted on nucleic acids. Data on the enzyme systemsWhatman 1 filter paper sheets. According to the participating in these metabolic processes arereaction products expected, a choice was made of rather fragmentary (1—9) and some are, inat least 2 among the following solvents, all used terms of biochemical time, in need of up- in ascending direction: 1. isoamyl alcohol—5% Na2HPO4 (1:1), dating. In some other publications (10—18), 2. water-saturated n-butanol, the presence and concentration of various in- 3. distilled water, termediate products is given. 4. 80% formic acid—n-hutanol——n-propanol— In this paper, we tried to collect and supple- acetone—30% trichloro-aeetic acid (5:8:4: ment these data by investigating the presence 5:3), 5. n-butanol——4% boric acid (43:7), or absence in epidermis of enzyme systems 6. isobutyrie acid—water—ammonia 0.880—ver- that have been described in other tissues. sene 0.1M(500:279:21:8), This first investigation was a qualitative one, 7. upper phase of ethyl acetate—water—formic and some limitations were set by practical acid (12:7:1), 8.
    [Show full text]
  • From Helicobacter Pylori
    Biochemical Characterization of Hypothetical Proteins from Helicobacter pylori Han-Pil Choi1, Silvia Juarez2, Sergio Ciordia2, Marisol Fernandez2, Rafael Bargiela3, Juan P. Albar2, Varun Mazumdar4, Brian P. Anton5, Simon Kasif1,4, Manuel Ferrer3*, Martin Steffen1,6* 1 Dept of Biomedical Engineering, Boston University, Boston, Massachusetts, United States of America, 2 Proteomic Facility, CNB-National Centre for Biotechnology, CSIC, Darwin 3, Madrid, Spain, 3 Spanish National Research Council (CSIC), Institute of Catalysis, Madrid, Spain, 4 Bioinformatics Program, Boston University, Boston, Massachusetts, United States of America, 5 New England Biolabs, Ipswich, Massachusetts, United States of America, 6 Dept of Pathology and Laboratory Medicine, Boston University School of Medicine, Boston, Massachusetts, United States of America Abstract The functional characterization of Open Reading Frames (ORFs) from sequenced genomes remains a bottleneck in our effort to understand microbial biology. In particular, the functional characterization of proteins with only remote sequence homology to known proteins can be challenging, as there may be few clues to guide initial experiments. Affinity enrichment of proteins from cell lysates, and a global perspective of protein function as provided by COMBREX, affords an approach to this problem. We present here the biochemical analysis of six proteins from Helicobacter pylori ATCC 26695, a focus organism in COMBREX. Initial hypotheses were based upon affinity capture of proteins from total cellular lysate
    [Show full text]
  • CRISPR-Cas9 DNA Base-Editing and Prime-Editing
    International Journal of Molecular Sciences Review CRISPR-Cas9 DNA Base-Editing and Prime-Editing Ariel Kantor 1,2,*, Michelle E. McClements 1,2 and Robert E. MacLaren 1,2 1 Nuffield Laboratory of Ophthalmology, Nuffield Department of Clinical Neurosciences & NIHR Oxford Biomedical Research Centre, University of Oxford, Oxford OX3 9DU, UK 2 Oxford Eye Hospital, Oxford University Hospitals NHS Foundation Trust, Oxford OX3 9DU, UK * Correspondence: [email protected] Received: 14 July 2020; Accepted: 25 August 2020; Published: 28 August 2020 Abstract: Many genetic diseases and undesirable traits are due to base-pair alterations in genomic DNA. Base-editing, the newest evolution of clustered regularly interspaced short palindromic repeats (CRISPR)-Cas-based technologies, can directly install point-mutations in cellular DNA without inducing a double-strand DNA break (DSB). Two classes of DNA base-editors have been described thus far, cytosine base-editors (CBEs) and adenine base-editors (ABEs). Recently, prime-editing (PE) has further expanded the CRISPR-base-edit toolkit to all twelve possible transition and transversion mutations, as well as small insertion or deletion mutations. Safe and efficient delivery of editing systems to target cells is one of the most paramount and challenging components for the therapeutic success of BEs. Due to its broad tropism, well-studied serotypes, and reduced immunogenicity, adeno-associated vector (AAV) has emerged as the leading platform for viral delivery of genome editing agents, including DNA-base-editors. In this review, we describe the development of various base-editors, assess their technical advantages and limitations, and discuss their therapeutic potential to treat debilitating human diseases.
    [Show full text]