Development of a recombinant fusion protein based on the dynein light chain LC8 for non-viral gene delivery

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. (C) 2012 Elsevier B.V. All rights reserved.Fundacao de Amparo a Pesquisa do Estado de Sao Paulo - FAPESP (Sao Paulo, Brazil)Laboratorio de Espectroscopia e Calorimetria (LEC), Laboratorio Nacional de Biociencias - LNBio (Campinas, Brazil

Functional and structural studies of the disulfide isomerase DsbC from the plant pathogen Xylella fastidiosa reveals a redox-dependent oligomeric modulation in vitro

Xylella fastidiosa is a Gram-negative bacterium that grows as a biofilm inside the xylem vessels of susceptible plants and causes several economically relevant crop diseases. In the present study, we report the functional and low-resolution structural characterization of the X. fastidiosa disulfide isomerase DsbC (XfDsbC). DsbC is part of the disulfide bond reduction/isomerization pathway in the bacterial periplasm and plays an important role in oxidative protein folding. In the present study, we demonstrate the presence of XfDsbC during different stages of X. fastidiosa biofilm development. XfDsbC was not detected during X. fastidiosa planktonic growth; however, after administering a sublethal copper shock, we observed an overexpression of XfDsbC that also occurred during planktonic growth. These results suggest that X. fastidiosa can use XfDsbC in vivo under oxidative stress conditions similar to those induced by copper. In addition, using dynamic light scattering and small-angle X-ray scattering, we observed that the oligomeric state of XfDsbC in vitro may be dependent on the redox environment. Under reducing conditions, XfDsbC is present as a dimer, whereas a putative tetrameric form was observed under nonreducing conditions. Taken together, our findings demonstrate the overexpression of XfDsbC during biofilm formation and provide the first structural model of a bacterial disulfide isomerase in solution. Structured digital abstract XfDsbC and XfDsbC bind by x ray scattering (View Interaction: 1, 2) XfDsbC and XfDsbC bind by molecular sieving (View interaction) XfDsbC and XfDsbC bind by comigration in non denaturing gel electrophoresis (View interaction) XfDsbC and XfDsbC bind by cross-linking study (View Interaction: 1, 2) XfDsbC and XfDsbC bind by dynamic light scattering (View Interaction: 1, 2)Fundacao de Amparo a Pesquisa do Estado de Sao Paulo (FAPESP)Fundacao de Amparo a Pesquisa do Estado de Sao Paulo (FAPESP) [Process 01/07533-7, Process 08/55690-3]Conselho Nacional de Desenvolvimento Cientifico e Tecnologico (CNPq)Conselho Nacional de Desenvolvimento Cientifico e Tecnologico (CNPq

Techno-Economic Analysis of a Hyaluronic Acid Production Process Utilizing Streptococcal Fermentation

Hyaluronic acid (HA) is a polysaccharide of alternating d-glucuronic acid and N-acetyl-d-glucosamine residues present in the extracellular matrix of connective, epithelial, and nervous tissues. Due to its singular hydrating, rheological and adhesive properties, HA has found numerous cosmetic and medical applications. However, techno-economic analyses of high value-added bioproducts such as HA are scarce in the literature. Here, we present a techno-economic analysis of a process for producing HA using Streptococcus zooepidemicus, simulated in SuperPro Designer. In the baseline scenario, HA is produced by batch fermentation, reaching 2.5 g/L after 24 h. It is then centrifuged, diafiltered, treated with activated carbon and precipitated with isopropanol. The product is suitable for topical formulations and its production cost was estimated as 1115 $/kg. A similar scenario, based on fed-batch culture and assuming a titer of 5.0 g/L, led to a lower cost of 946$/kg. Moreover, in two additional scenarios, 10% of the precipitated HA is diverted to the production of a highly pure and high-molecular weight HA, suitable for injectable applications. These scenarios resulted in higher capital and operating costs, but also in higher profits, because HA for injectable use has a higher selling price that more than compensates for its higher production costs

Recombinant protein-based nanocarriers and their association with cationic liposomes: Characterization and in vitro evaluation

A major bottleneck in the development of efficient protocols for gene therapy and DNA vaccination is the low efficiency of gene transfer by non-viral vectors. This is mainly attributed to the fact that, during the traffic to target cell nuclei, vectors must overcome a series of enzymatic, physical, and diffusional barriers. The objective of this work was the development and characterization of new multifunctional non-viral vectors, based on proteins and lipids, that are able to efficiently deliver the foreign plasmid DNA (pDNA) to the nucleus of mammalian cells. A model pDNA containing the reporter gene GFP was complexed to protamine or the recombinant modular protein T-Rp3 to form binary complexes. In addition, we studied the ability of the cationic liposome composed of EPC/DOPE/DOTAP to encapsulate the binary complexes to form pseudo-ternary complexes (pDNA/protein/liposome). Characterization of the complexes were performed by dynamic light scattering (DLS), zeta potential, transmission electron microscopy (TEM) and pDNA accessibility assay. The assays revealed that both proteins were able to condense pDNA and form positively charged complexes, that could be efficiently encapsulated, leading to the formation of pDNA/protein/liposome complexes. Transfection studies using HeLa cells indicated that pDNA/protein formed by T-Rp3 were far more efficient for pDNA delivery than protamine. The complexes formed by pDNA/T-Rp3/liposome presented the highest transfection level (25%). On the other hand, cytotoxicity assays showed a significant decrease on cell viability when using pDNA/T-Rp3/liposome, indicating that the association of T-Rp3 with liposome significantly increase the delivery efficiency whilst prompting a proportional negative impact on cytotoxicity. A better understanding of the mechanisms of cell uptake and intracellular trafficking regarding the synergic effect between proteins and lipids in these vectors may, in the near future, lead to the development of more efficient non-viral vectors able to mimic the abilities of viral nucleic acid delivery513110CONSELHO NACIONAL DE DESENVOLVIMENTO CIENTÍFICO E TECNOLÓGICO - CNPQFUNDAÇÃO DE AMPARO À PESQUISA DO ESTADO DE SÃO PAULO - FAPESP471971/2011-1; 444412/2014-02007/58323-9; 2013/23780-1; 2012/23143-9; 2013/05969-

Characterization Of The Tolb-pal Trans-envelope Complex From Xylella Fastidiosa Reveals A Dynamic And Coordinated Protein Expression Profile During The Biofilm Development Process.

The intriguing roles of the bacterial Tol-Pal trans-envelope protein complex range from maintenance of cell envelope integrity to potential participation in the process of cell division. In this study, we report the characterization of the XfTolB and XfPal proteins of the Tol-Pal complex of Xylella fastidiosa. X. fastidiosa is a major plant pathogen that forms biofilms inside xylem vessels, triggering the development of diseases in important cultivable plants around the word. Based on functional complementation experiments in Escherichia coli tolB and pal mutant strains, we confirmed the role of xftolB and xfpal in outer membrane integrity. In addition, we observed a dynamic and coordinated protein expression profile during the X. fastidiosa biofilm development process. Using small-angle X-ray scattering (SAXS), the low-resolution structure of the isolated XfTolB-XfPal complex in solution was solved for the first time. Finally, the localization of the XfTolB and XfPal polar ends was visualized via immunofluorescence labeling in vivo during bacterial cell growth. Our results highlight the major role of the components of the cell envelope, particularly the TolB-Pal complex, during the different phases of bacterial biofilm development

Correlation of the physicochemical and structural properties of pDNA/cationic liposome complexes with their in vitro transfection

In this study, we characterized the conventional physicochemical properties of the complexes formed by plasmid DNA (pDNA) and cationic liposomes (CL) composed of egg phosphatidylcholine (EPC), 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), and 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP) (50/25/25% molar ratio). We found that these properties are nearly unaffected at the studied ranges when the molar charge ratio (R±) between the positive charge from the CL and negative charge from pDNA is not close to the isoneutrality region (R± = 1). However, the results from in vitro transfection of HeLa cells showed important differences when R± is varied, indicating that the relationships between the physicochemical and biological characteristics were not completely elucidated. To obtain information regarding possible liposome structural modifications, small-angle X-ray scattering (SAXS) experiments were performed as a function of R± to obtain correlations between structural, physicochemical, and transfection properties. The SAXS results revealed that pDNA/CL complexes can be described as being composed of single bilayers, double bilayers, and multiple bilayers, depending on the R± value. Interestingly, for R± = 9, 6, and 3, the system is composed of single and double bilayers, and the fraction of the latter increases with the amount of DNA (or a decreasing R±) in the system. This information is used to explain the transfection differences observed at an R± = 9 as compared to R± = 3 and 6. Close to the isoneutrality region (R± = 1.8), there was an excess of pDNA, which induced the formation of a fraction of aggregates with multiple bilayers. These aggregates likely provide additional resistance against the release of pDNA during the transfection phenomenon, reflected as a decrease in the transfection level. The obtained results permitted proper correlation of the physicochemical and structural properties of pDNA/CL complexes with the in vitro transfection of HeLa cells by these complexes, contributing to a better understanding of the gene delivery process28311153511545FUNDAÇÃO DE AMPARO À PESQUISA DO ESTADO DE SÃO PAULO - FAPESPsem informaçã

Development and characterization of a cationic lipid nanocarrier as non-viral vector for gene therapy

The aim of the present work was to produce a cationic solid lipid nanoparticle (SLN) as non-viral vector for protein delivery. Cationic SLN were produced by double emulsion method, composed of softisan® 100, cetyltrimethylammonium bromide (CTAB), Tween® 80, Span® 80, glycerol and lipoid® S75 loading insulin as model protein. The formulation was characterized in terms of mean hydrodynamic diameter (z-ave), polydispersity index (PI), zeta potential (ZP), stability during storage time, stability after lyophilization, effect of toxicity and transfection ability in HeLa cells, in vitro release profile and morphology. SLN were stable for 30 days and showed minimal changes in their physicochemical properties after lyophilization. The particles exhibited a relatively slow release, spherical morphology and were able to transfect HeLa cells, but toxicity remained an obstacle. Results suggest that SLN are nevertheless promising for delivery of proteins or nucleic acids for gene therapy667882COORDENAÇÃO DE APERFEIÇOAMENTO DE PESSOAL DE NÍVEL SUPERIOR - CAPESFUNDAÇÃO DE AMPARO À PESQUISA DO ESTADO DE SÃO PAULO - FAPESPsem informação2011/20801-2; 2008/00421-

Development And Characterization Of A Cationic Lipid Nanocarrier As Non-viral Vector For Gene Therapy.

The aim of the present work was to produce a cationic solid lipid nanoparticle (SLN) as non-viral vector for protein delivery. Cationic SLN were produced by double emulsion method, composed of softisan(®) 100, cetyltrimethylammonium bromide (CTAB), Tween(®) 80, Span(®) 80, glycerol and lipoid(®) S75 loading insulin as model protein. The formulation was characterized in terms of mean hydrodynamic diameter (z-ave), polydispersity index (PI), zeta potential (ZP), stability during storage time, stability after lyophilization, effect of toxicity and transfection ability in HeLa cells, in vitro release profile and morphology. SLN were stable for 30days and showed minimal changes in their physicochemical properties after lyophilization. The particles exhibited a relatively slow release, spherical morphology and were able to transfect HeLa cells, but toxicity remained an obstacle. Results suggest that SLN are nevertheless promising for delivery of proteins or nucleic acids for gene therapy.66C78-8

Correlation Of The Physicochemical And Structural Properties Of Pdna/cationic Liposome Complexes With Their In Vitro Transfection.

In this study, we characterized the conventional physicochemical properties of the complexes formed by plasmid DNA (pDNA) and cationic liposomes (CL) composed of egg phosphatidylcholine (EPC), 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), and 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP) (50/25/25% molar ratio). We found that these properties are nearly unaffected at the studied ranges when the molar charge ratio (R(±)) between the positive charge from the CL and negative charge from pDNA is not close to the isoneutrality region (R(±) = 1). However, the results from in vitro transfection of HeLa cells showed important differences when R(±) is varied, indicating that the relationships between the physicochemical and biological characteristics were not completely elucidated. To obtain information regarding possible liposome structural modifications, small-angle X-ray scattering (SAXS) experiments were performed as a function of R(±) to obtain correlations between structural, physicochemical, and transfection properties. The SAXS results revealed that pDNA/CL complexes can be described as being composed of single bilayers, double bilayers, and multiple bilayers, depending on the R(±) value. Interestingly, for R(±) = 9, 6, and 3, the system is composed of single and double bilayers, and the fraction of the latter increases with the amount of DNA (or a decreasing R(±)) in the system. This information is used to explain the transfection differences observed at an R(±) = 9 as compared to R(±) = 3 and 6. Close to the isoneutrality region (R(±) = 1.8), there was an excess of pDNA, which induced the formation of a fraction of aggregates with multiple bilayers. These aggregates likely provide additional resistance against the release of pDNA during the transfection phenomenon, reflected as a decrease in the transfection level. The obtained results permitted proper correlation of the physicochemical and structural properties of pDNA/CL complexes with the in vitro transfection of HeLa cells by these complexes, contributing to a better understanding of the gene delivery process.2811535-4

Correlation of the Physicochemical and Structural Properties of pDNA/Cationic Liposome Complexes with Their in Vitro Transfection

In this study, we characterized the conventional physicochemical properties of the complexes formed by plasmid DNA (pDNA) and cationic liposomes (CL) composed of egg phosphatidylcholine (EPC), 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), and 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP) (50/25/25% molar ratio). We found that these properties are nearly unaffected at the studied ranges when the molar charge ratio (R-+/-) between the positive charge from the CL and negative charge from pDNA is not close to the isoneutrality region (R-+/- = 1). However, the results from in vitro transfection of HeLa cells showed important differences when R-+/- is varied, indicating that the relationships between the physicochemical and biological characteristics were not completely elucidated. To obtain information regarding possible liposome structural modifications, small-angle X-ray scattering (SAXS) experiments were performed as a function of R-+/- to obtain correlations between structural, physicochemical, and transfection properties. The SAXS results revealed that pDNA/CL complexes can be described as being composed of single bilayers, double bilayers, and multiple bilayers, depending on the R-+/- value. Interestingly, for R-+/- = 9, 6, and 3, the system is composed of single and double bilayers, and the fraction of the latter increases with the amount of DNA (or a decreasing R-+/-) in the system. This information is used to explain the transfection differences observed at an R-+/- = 9 as compared to R-+/- = 3 and 6. Close to the isoneutrality region (R-+/- = 1.8), there was an excess of pDNA, which induced the formation of a fraction of aggregates with multiple bilayers. These aggregates likely provide additional resistance against the release of pDNA during the transfection phenomenon, reflected as a decrease in the transfection level. The obtained results permitted proper correlation of the physicochemical and structural properties of pDNA/CL complexes with the in vitro transfection of HeLa cells by these complexes, contributing to a better understanding of the gene delivery process.Fundacao de Amparo a Pesquisa do Estado de Sao Paulo - FAPESP (Sao Paulo, Brazil)Universidade Estadual de Campinas (Campinas, Brazil