Taming hyperactive hDNase I: Stable inducible expression of a hyperactive salt- and actin-resistant variant of human deoxyribonuclease I in CHO cells.

Item request has been placed! ×
Item request cannot be made. ×
loading   Processing Request
Read More Add to Saved list
  • Additional Information
    • Source:
      Publisher: Wiley-Blackwell Country of Publication: United States NLM ID: 8506292 Publication Model: Print-Electronic Cited Medium: Internet ISSN: 1520-6033 (Electronic) Linking ISSN: 15206033 NLM ISO Abbreviation: Biotechnol Prog Subsets: MEDLINE
    • Publication Information:
      Publication: <2010-> : Hoboken, NJ : Wiley-Blackwell
      Original Publication: [New York, N.Y. : American Institute of Chemical Engineers, c1985-
    • Subject Terms:
      Actins/*chemistry ; Cloning, Molecular/*methods ; Deoxyribonuclease I/*chemistry ; Deoxyribonuclease I/*metabolism ; Protein Engineering/*methods ; Salts/*chemistry; Animals ; CHO Cells ; Cell Proliferation/physiology ; Cricetulus ; Deoxyribonuclease I/genetics ; Enzyme Activation ; Enzyme Stability ; Humans ; Recombinant Proteins/chemistry ; Recombinant Proteins/metabolism
    • Abstract:
      While the most common causes of clonal instability are DNA copy number loss and silencing, toxicity of the expressed protein(s) may also induce clonal instability. Human DNase I (hDNase I) is used therapeutically for the treatment of cystic fibrosis (CF) and may have potential benefit for use in systemic lupus erythematosus (SLE). hDNase I is an endonuclease that catalyzes degradation of extracellular DNA and is inhibited by both salt and G-actin. Engineered versions of hDNase I, bearing multiple point mutations, which renders them Hyperactive, Salt- and Actin-Resistant (HSAR-hDNase I) have been developed previously. However, constitutive expression of HSAR-hDNase I enzymes has been very challenging and, despite considerable efforts and screening thousands of clones, no stable clone capable of constitutive expression had been obtained. Here, we developed a regulated expression system for stable expression of an HSAR-hDNase I in Chinese Hamster Ovary (CHO) cells. The HSAR-hDNase I clones were stable and, upon induction, expressed enzymatically functional protein. Our findings suggest that degradation of host's DNA mediated by HSAR-hDNase I during cell division is the likely cause of clonal instability observed in cells constitutively expressing this protein. Purified HSAR-hDNase I was both hyperactive and resistant to inhibition by salt and G-actin, resulting in an enzyme having ca. 10-fold greater specific activity and the potential to be a superior therapeutic agent to wild type (WT) hDNase I. Furthermore, the ability to regulate hDNase I expression has enabled process development improvements that achieve higher cell growth and product titers while maintaining product quality. © 2017 American Institute of Chemical Engineers Biotechnol. Prog., 32:523-533, 2017.
      (© 2017 American Institute of Chemical Engineers.)
    • Contributed Indexing:
      Keywords: cystic fibrosis; hDNase I; hyperactive; inducible expression
    • Accession Number:
      0 (Actins)
      0 (Recombinant Proteins)
      0 (Salts)
      EC 3.1.21.- (DNASE1L2 protein, human)
      EC 3.1.21.1 (Deoxyribonuclease I)
    • Publication Date:
      Date Created: 20170128 Date Completed: 20180104 Latest Revision: 20180204
    • Publication Date:
      20240829
    • Accession Number:
      10.1002/btpr.2439
    • Accession Number:
      28127892