title: Development of a recombinant fusion protein based on the dynein light chain LC8 for non-viral gene delivery.
abstract: The low efficiency of gene transfer is a recurrent problem in DNA vaccine development and gene therapy studies using non-viral vectors such as plasmid DNA (pDNA). This is mainly due to the fact that during their traffic to the target cell's nuclei, plasmid vectors must overcome a series of physical, enzymatic and diffusional barriers. The main objective of this work is the development of recombinant proteins specifically designed for pDNA delivery, which take advantage of molecular motors like dynein, for the transport of cargos from the periphery to the centrosome of mammalian cells. A DNA binding sequence was fused to the N-terminus of the recombinant human dynein light chain LC8. Expression studies indicated that the fusion protein was correctly expressed in soluble form using E. coli BL21(DE3) strain. As expected, gel permeation assays found the purified protein mainly present as dimers, the functional oligomeric state of LC8. Gel retardation assays and atomic force microscopy proved the ability of the fusion protein to interact and condense pDNA. Zeta potential measurements indicated that LC8 with DNA binding domain (LD4) has an enhanced capacity to interact and condense pDNA, generating positively charged complexes. Transfection of cultured HeLa cells confirmed the ability of the LD4 to facilitate pDNA uptake and indicate the involvement of the retrograde transport in the intracellular trafficking of pDNA:LD4 complexes. Finally, cytotoxicity studies demonstrated a very low toxicity of the fusion protein vector, indicating the potential for in vivo applications. The study presented here is part of an effort to develop new modular shuttle proteins able to take advantage of strategies used by viruses to infect mammalian cells, aiming to provide new tools for gene therapy and DNA vaccination studies.
authors.affiliation: Laboratory of Genetics and Molecular Analysis, Molecular Biology and Genetic Engineering Center, State University of Campinas, Campinas, SP, Brazil.
authors.forename: Marcelo A S
authors.initials: MA
authors.lastname: Toledo
authors.forename: Richard
authors.initials: R
authors.lastname: Janissen
authors.forename: Marianna T P
authors.initials: MT
authors.lastname: Favaro
authors.forename: Mônica A
authors.initials: MA
authors.lastname: Cotta
authors.forename: Gabriel A
authors.initials: GA
authors.lastname: Monteiro
authors.forename: Duarte Miguel F
authors.initials: DM
authors.lastname: Prazeres
authors.forename: Anete P
authors.initials: AP
authors.lastname: Souza
authors.forename: Adriano R
authors.initials: AR
authors.lastname: Azzoni
mesh_terms: D018929:Cell Culture Techniques; D002470:Cell Survival; D002850:Chromatography, Gel; D002942:Circular Dichroism; D003001:Cloning, Molecular; D056445:Cytoplasmic Dyneins / Q000737:chemistry* / Q000235:genetics; D004247:DNA / Q000008:administration & dosage* / Q000235:genetics; D004337:Drug Carriers / Q000737:chemistry; D024202:Electrophoretic Mobility Shift Assay; D004926:Escherichia coli / Q000235:genetics; D018014:Gene Transfer Techniques; D006367:HeLa Cells; D006801:Humans; D018625:Microscopy, Atomic Force; D010316:Particle Size; D010957:Plasmids / Q000008:administration & dosage* / Q000235:genetics; D011485:Protein Binding; D015202:Protein Engineering; D011993:Recombinant Fusion Proteins / Q000737:chemistry* / Q000235:genetics; D013499:Surface Properties
chemical_list: D004337:Drug Carriers; D011993:Recombinant Fusion Proteins; D004247:DNA; C427758:DYNLL1 protein, human; D056445:Cytoplasmic Dyneins