Characterization of the human dynein light chain Rp3 and its use as a non-viral gene delivery vector

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• 作者：M. A. S. Toledo (1)
  M. T. P. Favaro (1)
  R. F. Alves (3)
  C. A. Santos (1)
  L. L. Beloti (1)
  A. Crucello (1)
  A. S. Santiago (1)
  J. S. Mendes (1)
  M. A. C. Horta (1)
  R. Aparicio (2)
  A. P. Souza (1)
  A. R. Azzoni (3)
  
• 关键词：Rp3 ; Dynein ; Gene delivery ; SAXS ; Transfection
• 刊名：Applied Microbiology and Biotechnology
• 出版年：2014
• 出版时间：April 2014
• 年：2014
• 卷：98
• 期：8
• 页码：3591-3602
• 全文大小：1,803 KB
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• 作者单位：M. A. S. Toledo (1)
  M. T. P. Favaro (1)
  R. F. Alves (3)
  C. A. Santos (1)
  L. L. Beloti (1)
  A. Crucello (1)
  A. S. Santiago (1)
  J. S. Mendes (1)
  M. A. C. Horta (1)
  R. Aparicio (2)
  A. P. Souza (1)
  A. R. Azzoni (3)
  
  1. Centro de Biologia Molecular e Engenharia Gen茅tica, Universidade Estadual de Campinas, Campinas, SP, Brazil
  3. Departamento de Engenharia Qu铆mica, Escola Polit茅cnica, Universidade de S茫o Paulo, Av. Prof. Luciano Gualberto, Trav. 3, N潞 380, 05508-900, S茫o Paulo, SP, Brazil
  2. Laborat贸rio de Biologia Estrutural e Cristalografia, Instituto de Qu铆mica, Universidade Estadual de Campinas, Campinas, SP, Brazil
  
• ISSN：1432-0614

文摘

Dynein light chains mediate the interaction between the cargo and the dynein motor complex during retrograde microtubule-mediated transport in eukaryotic cells. In this study, we expressed and characterized the recombinant human dynein light chain Rp3 and developed a modified variant harboring an N-terminal DNA-binding domain (Rp3-Db). Our approach aimed to explore the retrograde cell machinery based on dynein to enhance plasmid DNA (pDNA) traffic along the cytosol toward the nucleus. In the context of non-viral gene delivery, Rp3-Db is expected to simultaneously interact with DNA and dynein, thereby enabling a more rapid and efficient transport of the genetic material across the cytoplasm. We successfully purified recombinant Rp3 and obtained a low-resolution structural model using small-angle X-ray scattering. Additionally, we observed that Rp3 is a homodimer under reducing conditions and remains stable over a broad pH range. The ability of Rp3 to interact with the dynein intermediate chain in vitro was also observed, indicating that the recombinant Rp3 is correctly folded and functional. Finally, Rp3-Db was successfully expressed and purified and exhibited the ability to interact with pDNA and mediate the transfection of cultured HeLa cells. Rp3-Db was also capable of interacting in vitro with dynein intermediate chains, indicating that the addition of the N-terminal DNA-binding domain does not compromise its function. The transfection level observed for Rp3-Db is far superior than that reported for protamine and is comparable to that of the cationic lipid LipofectamineTM. This report presents an initial characterization of a non-viral delivery vector based on the dynein light chain Rp3 and demonstrates the potential use of modified human light chains as gene delivery vectors.