An improved low-endotoxin Clean Genome E. coli strain for production of biological products
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Principal Investigator: FREDERICK R BLATTNER Organization: SCARAB GENOMICS, LLC Fiscal Year: 2024 Award: $842,151 Funding agency: National Institute of General Medical Sciences Abstract E. coli has played a vital role in the production of recombinant proteins and plasmid DNA (pDNA) for therapeutic use since the advent of the biopharmaceutical industry. E. coli remains the workhorse in the production of heterologous proteins and pDNA because of its short doubling time, its ability to grow to high cell densities and its relatively straightforward scale-up potential. About 30% of all approved therapeutic proteins and 70% of anti- cancer agents continue to be made in E. coli. As the need for high quality pDNA has increased due to success in gene therapy and mRNA vaccines, there is a growing need for high quality, low toxicity bacterial strains for production of plasmids. The goal of this direct to Phase II application is to develop a versatile next-generation E. coli strain built using Scarab Genomics’ Clean Genome® platform that combines low endotoxin levels with enhanced genetic stability characteristics for nucleic acid and protein production. Specifically, we propose to use genetic techniques to substantially lower the level of contaminating endotoxin in pDNA and recombinant protein preparations. Scarab Genomics has developed and patented its reduced genome E. coli strains, which has removed 650 potentially contaminating proteins and all transposable elements. We propose to further eliminate genes that are responsible for the activation of the Toll-like receptor-4 (TLR-4). We propose that phosphorylation sites on lipid A and on carbohydrates on the LPS side chains activate the Limulus amebocyte lysate (LAL) assay. These will be removed and pDNA and protein products from this strain tested in a TLR-4 and LAL assay. Success of the proposal will culminate in an E. coli strain that is low in endotoxin and increased genetic stability but continues to generate high levels of nucleic acid and protein under fermentation conditions. Terms: <Address><Anti-Cancer Agents><Antibody Fragments><Antineoplastic Agents><Antineoplastic Drugs><Antineoplastics><Assay><Bacteria><Bioassay><Biological Agent><Biological Assay><Biological Products><Biotech><Biotechnology><Cancer Drug><Carbohydrates><Cell Density><Characteristics><Chimera Protein><Chimeric Proteins><Coupled><DNA><DNA Therapy><DNA Transposable Elements><DNA purification><Data><Denileukin Diftitox><Deoxyribonucleic Acid><Disease><Disorder><Drug Industry><E coli><E. coli><Elements><Encapsulated><Endotoxins><Engineering><Enzyme Gene><Enzymes><Escherichia coli><FDA approved><Fermentation><Fusion Protein><Gene Transfer Clinical><Generalized Growth><Genes><Genetic><Genetic Enhancement><Genetic Intervention><Genetic Technics><Genetic Techniques><Genome><Genomics><Goals><Growth><HPV 16><HPV-16><HPV16><Homolog of Drosophila TOLL><Human papilloma virus type 16><Human papillomavirus 16><Human papillomavirus type 16><IS Elements><Immunoglobulin Fragments><Insertion Sequence Elements><Interleukin-2 Fusion Protein><Interleukin-2 Fusion Toxin><Legal patent><Limulus><Lipid A><Measures><Methods><Neoplastic Disease Chemotherapeutic Agents><Nucleic Acids><Ontak><Patents><Phagocytes><Phagocytic Cell><Pharmaceutic Industry><Pharmaceutical Industry><Phase><Phosphatases><Phosphates><Phosphohydrolases><Phosphomonoesterases><Phosphoric Monoester Hydrolases><Phosphorylation><Phosphorylation Site><Plant Resins><Plasmids><Play><Preparation><Process><Production><Protein Phosphorylation><Proteins><Protocol><Protocols documentation><Publishing><RNA vaccine><RNA-based vaccine><Reagent><Receptor Protein><Recombinant Proteins><Research><Role><Safety><Salts><Sampling><Side><TLR4><TLR4 gene><Testing><Therapeutic><Therapeutic Uses><Time><Tissue Growth><Toll Homologue><Toxic effect><Toxicities><Transposable Elements><Tumor-Specific Treatment Agents><amebocyte><anti-cancer drug><biologics><biopharmaceutical><biopharmaceutical company><biopharmaceutical industry><biotherapeutic agent><comparative><cost><density><flasks><gene repair therapy><gene therapy><gene-based therapy><genetic therapy><genomic therapy><human papilloma virus 16><improved><inorganic phosphate><interest><mRNA vaccine><mRNA-based vaccine><native protein drug><next generation><ontogeny><pharmaceutical protein><plasmid DNA><preparations><protein drug agent><protein purification><protein-based drug><receptor><resin><scale up><small molecule><social role><success><therapeutic protein><toll-like receptor 4><type 16 Human papilloma virus><type 16 Human papillomavirus>