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

dc.creatorToledo, M
dc.creatorFavaro, MTP
dc.creatorAlves, RF
dc.creatorSantos, CA
dc.creatorBeloti, LL
dc.creatorCrucello, A
dc.creatorSantiago, AS
dc.creatorMendes, J
dc.creatorHorta, MAC
dc.creatorAparicio, R
dc.creatorSouza, AP
dc.creatorAzzoni, AR
dc.date2014
dc.dateAPR
dc.date2014-07-30T16:52:01Z
dc.date2015-11-26T16:37:17Z
dc.date2014-07-30T16:52:01Z
dc.date2015-11-26T16:37:17Z
dc.date.accessioned2018-03-28T23:20:22Z
dc.date.available2018-03-28T23:20:22Z
dc.identifierApplied Microbiology And Biotechnology. Springer, v. 98, n. 8, n. 3591, n. 3602, 2014.
dc.identifier0175-7598
dc.identifier1432-0614
dc.identifierWOS:000334171100018
dc.identifier10.1007/s00253-013-5239-5
dc.identifierhttp://www.repositorio.unicamp.br/jspui/handle/REPOSIP/62902
dc.identifierhttp://repositorio.unicamp.br/jspui/handle/REPOSIP/62902
dc.identifier.urihttp://repositorioslatinoamericanos.uchile.cl/handle/2250/1271994
dc.descriptionDynein 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 Lipofectamine(TM). 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.
dc.description98
dc.description8
dc.description3591
dc.description3602
dc.languageen
dc.publisherSpringer
dc.publisherNew York
dc.publisherEUA
dc.relationApplied Microbiology And Biotechnology
dc.relationAppl. Microbiol. Biotechnol.
dc.rightsfechado
dc.rightshttp://www.springer.com/open+access/authors+rights?SGWID=0-176704-12-683201-0
dc.sourceWeb of Science
dc.subjectRp3
dc.subjectDynein
dc.subjectGene delivery
dc.subjectSAXS
dc.subjectTransfection
dc.subjectX-ray-scattering
dc.subjectSmall-angle Scattering
dc.subjectCytoplasmic Dynein
dc.subjectBiological Macromolecules
dc.subjectSubunit Heterogeneity
dc.subjectIntermediate Chains
dc.subjectProtein-structure
dc.subjectPlasmid Dna
dc.subjectI-tasser
dc.subjectTctex-1
dc.titleCharacterization of the human dynein light chain Rp3 and its use as a non-viral gene delivery vector
dc.typeArtículos de revistas


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