DOCSLIB.ORG

Systems Biology Greatly Improve Activity of Secreted Therapeutic Sulfatase in CHO Bioprocess

Total Page：16

File Type：pdf, Size：1020Kb

Download full-text PDF Read full-text
Systems Biology Greatly Improve Activity of Secreted Therapeutic Sulfatase in CHO Bioprocess Systems biology greatly improve activity of secreted therapeutic sulfatase in CHO bioprocess Niklas Thalén1, Mona Moradi Barzadd1, Magnus Lundqvist1, Johanna Rodhe3, Monica Andersson3, Gholamreza Bidkhori2,4, Dominik Possner3, Chao Su3, Joakim Nilsson3, Peter Eisenhut5,6, Magdalena Malm1, Jeanette Westin3, Johan Forsberg3, Erik Nordling3, Adil Mardinoglu2, Anna-Luisa Volk1, Anna Sandegren3, Johan Rockberg1,* 1 Dept. of Protein science; KTH - Royal Institute of Technology; Stockholm; SE-106 91; Sweden 2 Science for Life Laboratory; KTH - Royal Institute of Technology; Solna; 171 65; Sweden 3 SOBI AB, Tomtebodavägen 23A, Stockholm, Sweden 4 AIVIVO Ltd. Unit 25, Bio-innovation centre, Cambridge Science park, Cambridge, UK. 5 ACIB - Austrian Centre of Industrial Biotechnology, Krenngasse 37, 8010 Graz, Austria 6 BOKU - University of Natural Resources and Life Sciences, Department of Biotechnology, Vienna, 1190, Austria * To whom correspondence should be addressed: Tel: +46 8 790 99 88; Email: [email protected] Target journal: Cell Systems Take home message: • Transcriptomic comparison of two CHO clones with different productivities showed three genes relevant for sulfatase activation and secretion • Co-expression of genes with sulfatase led to a 150-fold increase in specific activity • Reduced promoter strength increased specific activity of sulfatase SUMMARY Rare diseases are, despite their name, collectively common and millions of people are affected daily of conditions where treatment often is unavailable. Sulfatases are a large family of activating enzymes related to several of these diseases. Heritable genetic variations in sulfatases may lead to impaired activity and a reduced macromolecular breakdown within the lysosome, with several severe and lethal conditions as a consequence. While therapeutic options are scarce, treatment for some sulfatase deficiencies by recombinant enzyme replacement are available. However, such recombinant production of sulfatases suffers greatly from low product activity and yield, further limiting accessibility for patient groups. Here, we have addressed this problem by defining key-proteins necessary for active sulfatase secretion by comparison of CHO clones with different levels of production of active sulfatase. Quantitative transcriptomic analysis highlighted 14 key genes associated with sulfatase production, and experimental validation by co-expression improved the sulfatase enzyme activity by up to 150-fold. Furthermore, a correlation between product mRNA levels and sulfatase activity were observed and expression with lower activity promoters showed an increased in sulfatase activity. The workflow devised is general and we propose it to be useful for resolving bottlenecks in cellular machineries for improvement of cell factories for other biologics as well. INTRODUCTION Rare diseases were for a long time overseen by the pharmaceutical industry. With conditions that only affect a small portion of the public it was seen as not financially feasible to pursuit medical discoveries due to the low impact it would have. However, a shift took place three decades ago when country level initiatives (Gammie, Lu, & Ud-Din Babar, 2015) and rare disease communities (Shore et al., 2006; Zimmer, 2013) pushed for advancements in orphan drug development, ultimately leading to a drastic change within the industry, through regulatory incentives. And today, orphan drugs are a large player on the pharmaceutical market with 906 U.S. Food and Drug Administration (FDA) approved orphan drugs, as of September 2020 (U.S. Food and Drug administration, 2020) and an estimated market share of US $209 billion in 2022, accounting for a 21% of total branded prescription drug sales (Hadjivasiliou, 2017). Lysosomal storage disease (LSD) is a group of 50-60 rare metabolic disorders with a combined prevalence of 1 in every 2000 – 5000 live births. The diseases are characterized by an abnormal build-up of undigested molecules within the lysosome due to deficiency in one or several enzymes involved in the catabolic process (Mehta, Beck, & Sunder- Plassmann, 2006), leading to several severe and lethal conditions. Treatments for these rare conditions are often symptomatic and supportive but for some, the deficient enzyme can be replaced through infusion of a functional replacement. This enzyme replacement therapy (ERT) is strictly dependent on the production of highly purified and functional protein, as well as the effectiveness of their systemic distribution (Martino et al., 2005; Ries, 2017). Due to the importance of functional replacements all available ERT products needs to attain the essential glycans for uptake and transport to the lysosome (Muenzer et al., 2006). Sulfatases belong to a large conserved family of enzymes that are of particular interest for ERT since members from this enzyme family are deficient in seven different LSDs. There are 17 known human sulfatases that all share the same post-translational processing through the Endoplasmic reticulum (ER) and Golgi apparatus were post translational modifications (PTM) and activations takes place for correct function of the enzymes. Within the ER an activation process unique for sulfatases takes place where a C-alpha- formylglycin (FGly) is generated from a cysteine in the active site (Sardiello, Annunziata, Roma, & Ballabio, 2005b). This activation process is enabled through the formylglycine generating enzyme (FGE) that is encoded by the Sulfatase-modifying factor 1 (SUMF1) gene. This gene is crucial for sulfatase activation and was found through one of the more severe cases of LSD, the multiple sulfatase deficiency, were a mutation in SUMF1 leads to deficiencies in all sulfatases (Cosma et al., 2003b). Currently, three LSD’s, caused by sulfatase deficiencies, have ERT’s available (Idursulfase, Galsulfase, and Elosulfase alfa). Understanding the biology behind sulfatase assembly within the cell could improve not only our understanding of the processes that takes place for an important therapeutic target but it could also improve the production process for some of the most expensive pharmaceuticals on the market (Luzzatto et al., 2018). Chinese hamster ovary (CHO) cells have for a long time been the host of choice for production of various pharmaceuticals, and is today engineered into a sophisticated platform for production of high-quality products (Walsh, 2018). Improvements in CHO platform development has steadily increased its ability to produce high titers of a broad range of pharmaceuticals (Amann, Schmieder, Faustrup Kildegaard, Borth, & Andersen, 2019; Fischer, Handrick, & Otte, 2015; Wurm, 2004). These improvements have mainly been achieved through bioprocess optimization (Jayapal, Wlaschin, Hu, & Yap, 2007), were media composition and bioreactor design modifications have evolved. Selection of new production cell lines has traditionally been done through development and screening of a large number of mutated cells, out of which single clones are selected based on their capabilities to produce correct products, with no further directed development of the per cell yield. However, with the introduction of omics-based approaches to investigate the relationship of gene expressions and productivity levels, new frontiers for precise cell engineering are now available (Kildegaard, Baycin-Hizal, Lewis, & Betenbaugh, 2013; Kuo et al., 2018). Studies exploring gene level expressions and high productivity have found several important factors for increased yield at cell levels, opening up new possibilities for increasing the capabilities of CHO cell production of both new advanced biologics that require complex modifications as well as further titer increase of more traditional monoclonal antibodies (Hong, Lakshmanan, Goudar, & Lee, 2018). Currently, human derived cell lines or CHO cell lines are industry standards for production of enzymes for ERT in LSD due to the importance of correct glycosylation for efficient uptake of the drug (Tian et al., 2019; Whiteman & Kimura, 2017). In this study, sulfatase production is addressed through systems biology in order to find genes and processes involved in the production and secretion of active sulfatases. Two different sulfatases, N-sulphoglucosamine sulphohydrolase (Sulfamidase) and Arylsulfatase A (ASA), were included in order to identify transcriptomic components with impact on productivity regardless of sulfatase to be expressed. Quantitative transcriptomic comparison of two CHO cells with different sulfatase productivity were compared and three different genes related to sulfatase production were identified. Co-transfections of the identified genes yielded an increase from virtually undetectable activity up to a significant amount of sulfatase activity, showing the high impact omics studies can have on improvements of production of important difficult to express proteins. Also, expressing ASA with lower promoter activity through different promoter cassettes increased the specific activity of the enzyme. RESULTS Automated bioreactor Cultivation of CHO Cells with varying sulfatase produCtivities enables quantitative transCriptomiC analyses In order to study differential gene expression related to activation and production of sulfatases, sets of comparable CHO cultures are needed. Here, two CHO production cell clones expressing Sulfamidase were analyzed in a transcriptomics workflow (figure 1). Duplicate samples of the two Sulfamidase producing clones were cultivated for 17 days under three different conditions (standard
Load more

Recommended publications
  • RNA Editing at Baseline and Following Endoplasmic Reticulum Stress
    RNA Editing at Baseline and Following Endoplasmic Reticulum Stress By Allison Leigh Richards A dissertation submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy (Human Genetics) in The University of Michigan 2015 Doctoral Committee: Professor Vivian G. Cheung, Chair Assistant Professor Santhi K. Ganesh Professor David Ginsburg Professor Daniel J. Klionsky Dedication To my father, mother, and Matt without whom I would never have made it ii Acknowledgements Thank you first and foremost to my dissertation mentor, Dr. Vivian Cheung. I have learned so much from you over the past several years including presentation skills such as never sighing and never saying “as you can see…” You have taught me how to think outside the box and how to create and explain my story to others. I would not be where I am today without your help and guidance. Thank you to the members of my dissertation committee (Drs. Santhi Ganesh, David Ginsburg and Daniel Klionsky) for all of your advice and support. I would also like to thank the entire Human Genetics Program, and especially JoAnn Sekiguchi and Karen Grahl, for welcoming me to the University of Michigan and making my transition so much easier. Thank you to Michael Boehnke and the Genome Science Training Program for supporting my work. A very special thank you to all of the members of the Cheung lab, past and present. Thank you to Xiaorong Wang for all of your help from the bench to advice on my career. Thank you to Zhengwei Zhu who has helped me immensely throughout my thesis even through my panic.
    [Show full text]
  • Mammalian Protein Expression and Characterization Tools for Next Generation Biologics
    Mammalian protein expression and characterization tools for next generation biologics next generation for tools characterization and expression protein Mammalian Doctoral Thesis in Biotechnology Mammalian protein expression and characterization tools for next generation biologics NIKLAS THALÉN ISBN TRITACBHFOU: KTH www.kth.se Stockholm, Sweden Mammalian protein expression and characterization tools for next generation biologics NIKLAS THALÉN Academic Dissertation which, with due permission of the KTH Royal Institute of Technology, is submitted for public defence for the Degree of Doctor of Philosophy on June 11th, 2021 at 10:00, F3, Lindstedsvägen 26, våningsplan 2, Sing-Sing, KTH campus, Stockholm. Doctoral Thesis in Biotechnology KTH Royal Institute of Technology Stockholm, Sweden 2021 © Niklas Thalén ISBN 978-91-7873-927-1 TRITA-CBH-FOU-2021:21 Printed by: Universitetsservice US-AB, Sweden 2021 Till Sofia Abstract Protein therapeutics are increasingly important for modern medicine. Novel recombinant proteins developed today can bind towards their target with high specificity and with low adverse effect. This has enabled the treatment of diseases that for a few years ago were deemed uncurable. Discovery of therapeutic proteins is driven through protein engineering, a field that is in constant expansion. And, through artificial construction of recombinant proteins, a large array of diseases can be defeated. The function and quality of these protein therapeutics rely on the correct folding, assembly and residue modification that occurs during their production within a living production cell host. Furthermore, producing them in large quantities are essential for accessibility of the best biopharmaceuticals available. Commonly, mammalian cells are the production host of choice when it comes to production of biopharmaceuticals.
    [Show full text]
  • The Activation of Arylsulfatases
    The activation of arylsulfatases D'Eustachio, P., Jassal, B. European Bioinformatics Institute, New York University Langone Medical Center, Ontario Institute for Cancer Research, Oregon Health and Science University. The contents of this document may be freely copied and distributed in any media, provided the authors, plus the institutions, are credited, as stated under the terms of Creative Commons Attribution 4.0 Inter- national (CC BY 4.0) License. For more information see our license. 04/06/2019 Introduction Reactome is open-source, open access, manually curated and peer-reviewed pathway database. Pathway annotations are authored by expert biologists, in collaboration with Reactome editorial staff and cross- referenced to many bioinformatics databases. A system of evidence tracking ensures that all assertions are backed up by the primary literature. Reactome is used by clinicians, geneticists, genomics research- ers, and molecular biologists to interpret the results of high-throughput experimental studies, by bioin- formaticians seeking to develop novel algorithms for mining knowledge from genomic studies, and by systems biologists building predictive models of normal and disease variant pathways. The development of Reactome is supported by grants from the US National Institutes of Health (P41 HG003751), University of Toronto (CFREF Medicine by Design), European Union (EU STRP, EMI-CD), and the European Molecular Biology Laboratory (EBI Industry program). Literature references Fabregat, A., Sidiropoulos, K., Viteri, G., Forner, O., Marin-Garcia, P., Arnau, V. et al. (2017). Reactome pathway ana- lysis: a high-performance in-memory approach. BMC bioinformatics, 18, 142. ↗ Sidiropoulos, K., Viteri, G., Sevilla, C., Jupe, S., Webber, M., Orlic-Milacic, M. et al.
    [Show full text]
  • Variation in Protein Coding Genes Identifies Information Flow
    bioRxiv preprint doi: https://doi.org/10.1101/679456; this version posted June 21, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. Animal complexity and information flow 1 1 2 3 4 5 Variation in protein coding genes identifies information flow as a contributor to 6 animal complexity 7 8 Jack Dean, Daniela Lopes Cardoso and Colin Sharpe* 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 Institute of Biological and Biomedical Sciences 25 School of Biological Science 26 University of Portsmouth, 27 Portsmouth, UK 28 PO16 7YH 29 30 * Author for correspondence 31 [email protected] 32 33 Orcid numbers: 34 DLC: 0000-0003-2683-1745 35 CS: 0000-0002-5022-0840 36 37 38 39 40 41 42 43 44 45 46 47 48 49 Abstract bioRxiv preprint doi: https://doi.org/10.1101/679456; this version posted June 21, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. Animal complexity and information flow 2 1 Across the metazoans there is a trend towards greater organismal complexity. How 2 complexity is generated, however, is uncertain. Since C.elegans and humans have 3 approximately the same number of genes, the explanation will depend on how genes are 4 used, rather than their absolute number.
    [Show full text]
  • Investigating Host Genes Involved In. HIY Control by a Novel Computational Method to Combine GWAS with Eqtl
    Investigating Host Genes Involved in. HIY Control by a Novel Computational Method to Combine GWAS with eQTL by Yi Song THESIS Submitted In partial satisfaction of me teqoitements for the degree of MASTER OF SCIENCE In Biological and Medical Informatics In the GRADUATE DIVISION Copyright (2012) by Yi Song ii Acknowledgement First and foremost, I would like to thank my advisor Professor Hao Li, without whom this thesis would not have been possible. I am very grateful that Professor Li lead me into the field of human genomics and gave me the opportunity to pursue this interesting study in his laboratory. Besides the wealth of knowledge and invaluable insights that he offered in every meeting we had, Professor Li is one of the most approachable faculties I have met. I truly appreciate his patient guidance and his enthusiastic supervision throughout my master’s career. I am sincerely thankful to Professor Patricia Babbitt, the Associate Director of the Biomedical Informatics program at UCSF. Over my two years at UCSF, she has always been there to offer her help when I was faced with difficulties. I would also like to thank both Professor Babbitt and Professor Nevan Krogan for investing their valuable time in evaluating my work. I take immense pleasure in thanking my co-workers Dr. Xin He and Christopher Fuller. It has been a true enjoyment to discuss science with Dr. He, whose enthusiasm is a great inspiration to me. I also appreciate his careful editing of my thesis. Christopher Fuller, a PhD candidate in the Biomedical Informatics program, has provided great help for me on technical problems.
    [Show full text]
  • High-Resolution Genomic Copy Number Profiling of Glioblastoma Multiforme by Single Nucleotide Polymorphism DNA Microarray
    Published OnlineFirst May 12, 2009; DOI: 10.1158/1541-7786.MCR-08-0270 Published Online First on May 12, 2009 High-Resolution Genomic Copy Number Profiling of Glioblastoma Multiforme by Single Nucleotide Polymorphism DNA Microarray Dong Yin,1 Seishi Ogawa,3 Norihiko Kawamata,1 Patrizia Tunici,2 Gaetano Finocchiaro,4 Marica Eoli,4 Christian Ruckert,6 Thien Huynh,1 Gentao Liu,2 Motohiro Kato,3 Masashi Sanada,3 Anna Jauch,5 Martin Dugas,6 Keith L. Black,2 and H. Phillip Koeffler1 1Division of Hematology/Oncology and 2Maxine Dunitz Neurosurgical Institute, Cedars-Sinai Medical Center, University of California at Los Angeles School of Medicine, Los Angeles, California; 3Regeneration Medicine of Hematopoiesis, University of Tokyo, School of Medicine, Tokyo, Japan; 4National Neurological Institute “C Besta,” Milan, Italy; 5Institute of Human Genetics, University Hospital Heidelberg, Germany; and 6Department of Medical Informatics and Biomathematics, University of Munster, Munster, Germany Abstract growth factor receptor/platelet-derived growth factor receptor Glioblastoma multiforme (GBM) is an extremely malignant α. Deletion of chromosome 6q26-27 often occurred (16 of 55 brain tumor. To identify new genomic alterations in GBM, samples). The minimum common deleted region included genomic DNA of tumor tissue/explants from 55 individuals PARK2, PACRG, QKI,and PDE10A genes. Further reverse and 6 GBM cell lines were examined using single nucleotide transcription Q-PCR studies showed that PARK2 expression polymorphism DNA microarray (SNP-Chip). Further gene was decreased in another collection of GBMs at a expression analysis relied on an additional 56 GBM samples. frequency of 61% (34 of 56) of samples. The 1p36.23 region SNP-Chip results were validated using several techniques, was deleted in 35% (19 of 55) of samples.
    [Show full text]
  • SUMF2 Polyclonal Antibody (A01)
    SUMF2 polyclonal antibody (A01) Catalog # : H00025870-A01 規格 : [ 50 uL ] List All Specification Application Image Product Mouse polyclonal antibody raised against a partial recombinant SUMF2. Western Blot (Cell lysate) Description: Immunogen: SUMF2 (NP_056226, 26 a.a. ~ 125 a.a) partial recombinant protein with GST tag. Sequence: QATSMVQLQGGRFLMGTNSPDSRDGEGPVREATVKPFAIDIFPVTNKD FRDFVREKKYRTEAEMFGWSFVFEDFVSDELRNKATQPMKSVLWWLP enlarge VEKAF Western Blot (Recombinant protein) Host: Mouse ELISA Reactivity: Human Quality Control Antibody Reactive Against Recombinant Protein. Testing: Western Blot detection against Immunogen (37.11 KDa) . Storage Buffer: 50 % glycerol Storage Store at -20°C or lower. Aliquot to avoid repeated freezing and thawing. Instruction: MSDS: Download Datasheet: Download Applications Western Blot (Cell lysate) Page 1 of 2 2016/5/23 SUMF2 polyclonal antibody (A01), Lot # 060619JCS1 Western Blot analysis of SUMF2 expression in HepG2 ( Cat # L019V1 ). Protocol Download Western Blot (Recombinant protein) Protocol Download ELISA Gene Information Entrez GeneID: 25870 GeneBank NM_015411 Accession#: Protein NP_056226 Accession#: Gene Name: SUMF2 Gene Alias: DKFZp566I1024,DKFZp686I1024,DKFZp686L17160,DKFZp781L1035, MGC99485,pFGE Gene sulfatase modifying factor 2 Description: Omim ID: 607940 Gene Ontology: Hyperlink Gene Summary: The catalytic sites of sulfatases are only active if they contain a unique amino acid, C-alpha-formylglycine (FGly). The FGly residue is posttranslationally generated from a cysteine by enzymes with FGly- generating activity. The gene described in this record is a member of the sulfatase-modifying factor family and encodes a protein with a DUF323 domain that localizes to the lumen of the endoplasmic reticulum. This protein has low levels of FGly-generating activity but can heterodimerize with another family member - a protein with high levels of FGly-generating activity.
    [Show full text]
  • Mouse Sulfatase Modifying Factor 2/SUMF2 Antibody
    Mouse Sulfatase Modifying Factor 2/SUMF2 Antibody Monoclonal Rat IgG2A Clone # 382407 Catalog Number: MAB3454 DESCRIPTION Species Reactivity Mouse Specificity Detects mouse Sulfatase Modifying Factor 2/SUMF2 in direct ELISAs and Western blots. In Western blots, no cross­reactivity with recombinant human SUMF2 or recombinant mouse SUMF1 is observed. Source Monoclonal Rat IgG2A Clone # 382407 Purification Protein A or G purified from hybridoma culture supernatant Immunogen Mouse myeloma cell line NS0­derived recombinant mouse Sulfatase Modifying Factor 2/SUMF2 Gln34­Leu308 Accession # Q8BPG6 Formulation Lyophilized from a 0.2 μm filtered solution in PBS with Trehalose. See Certificate of Analysis for details. *Small pack size (­SP) is supplied as a 0.2 μm filtered solution in PBS. APPLICATIONS Please Note: Optimal dilutions should be determined by each laboratory for each application. General Protocols are available in the Technical Information section on our website. Recommended Sample Concentration Western Blot 1 µg/mL Recombinant Mouse Sulfatase Modifying Factor 2/SUMF2 PREPARATION AND STORAGE Reconstitution Reconstitute at 0.5 mg/mL in sterile PBS. Shipping The product is shipped at ambient temperature. Upon receipt, store it immediately at the temperature recommended below. *Small pack size (­SP) is shipped with polar packs. Upon receipt, store it immediately at ­20 to ­70 °C Stability & Storage Use a manual defrost freezer and avoid repeated freeze­thaw cycles. l 12 months from date of receipt, ­20 to ­70 °C as supplied. l 1 month, 2 to 8 °C under sterile conditions after reconstitution. l 6 months, ­20 to ­70 °C under sterile conditions after reconstitution. BACKGROUND Sulfatase Modifying Factor 2 (SUMF2) is structurally similar to SUMF1, which activates sulfatases by converting their active site residue cysteine to formylglycine.
    [Show full text]
  • Understanding Regulatory Mechanisms Underlying Stem Cells Helps to Identify Cancer Biomarkers
    Understanding Regulatory Mechanisms Underlying Stem Cells Helps to Identify Cancer Biomarkers A dissertation submitted towards the degree Doctor of Engineering (Dr.-Ing) of the Faculty of Mathematics and Computer Science of Saarland University by Maryam Nazarieh Saarbrücken, June 2018 i iii Day of Colloquium Jun 28, 2018 Dean of the Faculty Prof. Dr. Sebastian Hack Chair of the Committee Prof. Dr. Hans-Peter Lenhof Reporters First reviewer Prof. Dr. Volkhard Helms Second reviewer Prof. Dr. Dr. Thomas Lengauer Academic Assistant Dr. Christina Backes Acknowledgements Firstly, I would like to thank Prof. Volkhard Helms for offering me a position at his group and for his supervision and support on the SFB 1027 project. I am grateful to Prof. Thomas Lengauer for his helpful comments. I am thankful to Prof. Andreas Wiese for his contribution and discussion. I would like to thank Prof. Jan Baumbach that allowed me to spend a training phase in his group during my PhD preparatory phase and the collaborative work which I performed with his PhD student Rashid Ibragimov where I proposed a heuristic algorithm based on the characteristics of protein-protein interaction networks for solving the graph edit dis- tance problem. I would like to thank Graduate School of Computer Science and Center for Bioinformatics at Saarland University, especially Prof. Raimund Seidel and Dr. Michelle Carnell for giving me an opportunity to carry out my PhD studies. Furthermore, I would like to thank to Prof. Helms for enhancing my experience by intro- ducing master students and working as their advisor for successfully accomplishing their master projects.
    [Show full text]
  • Prion Characterization Using Cell Based Approaches
    University of Kentucky UKnowledge Theses and Dissertations--Microbiology, Microbiology, Immunology, and Molecular Immunology, and Molecular Genetics Genetics 2012 PRION CHARACTERIZATION USING CELL BASED APPROACHES Vadim Khaychuk University of Kentucky, [email protected] Right click to open a feedback form in a new tab to let us know how this document benefits ou.y Recommended Citation Khaychuk, Vadim, "PRION CHARACTERIZATION USING CELL BASED APPROACHES" (2012). Theses and Dissertations--Microbiology, Immunology, and Molecular Genetics. 2. https://uknowledge.uky.edu/microbio_etds/2 This Doctoral Dissertation is brought to you for free and open access by the Microbiology, Immunology, and Molecular Genetics at UKnowledge. It has been accepted for inclusion in Theses and Dissertations--Microbiology, Immunology, and Molecular Genetics by an authorized administrator of UKnowledge. For more information, please contact [email protected]. STUDENT AGREEMENT: I represent that my thesis or dissertation and abstract are my original work. Proper attribution has been given to all outside sources. I understand that I am solely responsible for obtaining any needed copyright permissions. I have obtained and attached hereto needed written permission statements(s) from the owner(s) of each third-party copyrighted matter to be included in my work, allowing electronic distribution (if such use is not permitted by the fair use doctrine). I hereby grant to The University of Kentucky and its agents the non-exclusive license to archive and make accessible my work in whole or in part in all forms of media, now or hereafter known. I agree that the document mentioned above may be made available immediately for worldwide access unless a preapproved embargo applies.
    [Show full text]
  • Table S1. 103 Ferroptosis-Related Genes Retrieved from the Genecards
    Table S1. 103 ferroptosis-related genes retrieved from the GeneCards. Gene Symbol Description Category GPX4 Glutathione Peroxidase 4 Protein Coding AIFM2 Apoptosis Inducing Factor Mitochondria Associated 2 Protein Coding TP53 Tumor Protein P53 Protein Coding ACSL4 Acyl-CoA Synthetase Long Chain Family Member 4 Protein Coding SLC7A11 Solute Carrier Family 7 Member 11 Protein Coding VDAC2 Voltage Dependent Anion Channel 2 Protein Coding VDAC3 Voltage Dependent Anion Channel 3 Protein Coding ATG5 Autophagy Related 5 Protein Coding ATG7 Autophagy Related 7 Protein Coding NCOA4 Nuclear Receptor Coactivator 4 Protein Coding HMOX1 Heme Oxygenase 1 Protein Coding SLC3A2 Solute Carrier Family 3 Member 2 Protein Coding ALOX15 Arachidonate 15-Lipoxygenase Protein Coding BECN1 Beclin 1 Protein Coding PRKAA1 Protein Kinase AMP-Activated Catalytic Subunit Alpha 1 Protein Coding SAT1 Spermidine/Spermine N1-Acetyltransferase 1 Protein Coding NF2 Neurofibromin 2 Protein Coding YAP1 Yes1 Associated Transcriptional Regulator Protein Coding FTH1 Ferritin Heavy Chain 1 Protein Coding TF Transferrin Protein Coding TFRC Transferrin Receptor Protein Coding FTL Ferritin Light Chain Protein Coding CYBB Cytochrome B-245 Beta Chain Protein Coding GSS Glutathione Synthetase Protein Coding CP Ceruloplasmin Protein Coding PRNP Prion Protein Protein Coding SLC11A2 Solute Carrier Family 11 Member 2 Protein Coding SLC40A1 Solute Carrier Family 40 Member 1 Protein Coding STEAP3 STEAP3 Metalloreductase Protein Coding ACSL1 Acyl-CoA Synthetase Long Chain Family Member 1 Protein
    [Show full text]
  • Anti-SUMF2 Polyclonal Antibody Cat: K108236P Summary
    Anti-SUMF2 Polyclonal Antibody Cat: K108236P Summary: 【Product name】: Anti-SUMF2 antibody 【Source】: Rabbit 【Isotype】: IgG 【Species reactivity】: Human Mouse Rat 【Swiss Prot】: Q8NBJ7 【Gene ID】: 25870 【Calculated】: MW:24/34/37kDa 【Observed】: MW:34kDa 【Purification】: Affinity purification 【Tested applications】: WB IHC 【Recommended dilution】: WB 1:1000-3000. IHC 1:50-200. 【WB Positive sample】: A549,HepG2,A431,K-562,THP-1,HMC-1,HT29,NIH3T3 【IHC Positive sample】: Human pancreatic cancer 【Subcellular location】: Cytoplasm 【Immunogen】: Recombinant protein of human SUMF2 【Storage】: Shipped at 4°C. Upon delivery aliquot and store at -20°C Background: The catalytic sites of sulfatases are only active if they contain a unique amino acid, C-alpha-formylglycine (FGly). The FGly residue is posttranslationally generated from a cysteine by enzymes with FGly-generating activity. The gene described in this record is a member of the sulfatase-modifying factor family and encodes a protein with a DUF323 domain that localizes to the lumen of the endoplasmic reticulum. This protein has low levels of FGly-generating activity but can heterodimerize with another family member - a protein with high levels of FGly-generating activity. Alternate transcriptional splice variants, encoding different isoforms, have been characterized. Sales:[email protected] For research purposes only. Tech:[email protected] Please visit www.solarbio.com for a more product information Verified picture Western blot analysis with SUMF2 antibody diluted at 1:2000;Lane: A549,HepG2,A431,K-562,THP-1,HMC-1,HT29,NIH3T3 Immunohistochemistry of paraffin-embedded Human pancreatic cancer with SUMF2 antibody diluted at 1:100 Sales:[email protected] For research purposes only.
    [Show full text]