Plasmid DNA partitioning and separation using poly(ethylene glycol)/poly(acrylate)/salt aqueous two-phase systems

Phase diagrams of poly(ethylene glycol)/polyacrylate/Na2SO4 systems have been investigated with respect to polymer size and pH. Plasmid DNA from Escherichia coil can depending on pH and polymer molecular weight be directed to a poly(ethylene glycol) or to a polyacrylate-rich phase in an aqueous two-phase system formed by these polymers. Bovine serum albumin (BSA) and E. coil homogenate proteins can be directed opposite to the plasmid partitioning in these systems. Two bioseparation processes have been developed where in the final step the pDNA is partitioned to a salt-rich phase giving a total process yield of 60-70%. In one of them the pDNA is partitioned between the polyacrylate and PEG-phases in order to remove proteins. In a more simplified process the plasmid is partitioned to a PEG-phase and back-extracted into a Na2SO4-rich phase. The novel polyacrylate/PEG system allows a strong change of the partitioning between the phases with relatively small changes in composition or pH. (C) 2012 Elsevier B.V. All rights reserved.

Generation and Biological Properties of a Recombinant Dodecahedron Containing the Short Fiber Protein of the Human Adenovirus 41

Objective: In order to gain further insight into the function of the enteric adenovirus short fiber (SF), we have constructed a recombinant dodecahedron containing the SF protein of HAdV-41 and the HAdV-3 penton base. Methods: Recombinant baculoviruses expressing the HAdV-41 SF protein and HAdV-3 penton base were cloned and amplified in Sf9 insect cells. Recombinant dodecahedra were expressed by coinfection of High Five (TM) cells with both baculoviruses, 72 h post-infection. Cell lysate was centrifuged on sucrose density gradient and the purified recombinant dodecahedra were recovered. Results: Analysis by negative staining electron microscopy demonstrated that chimeric dodecahedra made of the HAdV-3 penton base and decorated with the HAdV-41 SF were successfully generated. Next, recombinant dodecahedra were digested with pepsin and analyzed by Western blot. A 'site-specific' proteolysis of the HAdV-41 SF was observed, while the HAdV-3 penton base core was completely digested. Conclusion: These results show that, in vitro, the HAdV-41 SF likely undergoes proteolysis in the gastrointestinal tract, its natural environment, which may facilitate the recognition of receptors in intestinal cells. The results obtained in the present study may be the basis for the development of gene therapy vectors towards the intestinal epithelium, as well as orally administered vaccine vectors, but also for the HAdV-41 SF partner identification. Copyright (C) 2011 S. Karger AG, Basel

Recognition of enteroinvasive Escherichia coli and Shigella flexneri by dendritic cells: distinct dendritic cell activation states

The innate and adaptive immune responses of dendritic cells (DCs) to enteroinvasive Escherichia coli (EIEC) infection were compared with DC responses to Shigella flexneri infection. EIEC triggered DCs to produce interleukin (IL)-10, IL-12 and tumour necrosis factor (TNF)-alpha, whereas S. flexneri induced only the production of TNF-alpha. Unlike S. flexneri, EIEC strongly increased the expression of toll like receptor (TLR)-4 and TLR-5 in DCs and diminished the expression of co-stimulatory molecules that may cooperate to inhibit CD4(+) T-lymphocyte proliferation. The inflammation elicited by EIEC seems to be related to innate immunity both because of the aforementioned results and because only EIEC were able to stimulate DC transmigration across polarised Caco-2 cell monolayers, a mechanism likely to be associated with the secretion of CC chemokine ligands (CCL) 20 and TNF-alpha. Understanding intestinal DC biology is critical to unravelling the infection strategies of EIEC and may aid in the design of treatments for infectious diseases.

Production and characterization of poly-(3-hydroxybutyrate) from recombinant Escherichia coli grown on cheap renewable carbon substrates

Although the biopolymer poly-(3-hydroxybutyrate), P[3HB], presents physicochemical properties that make it an alternative material to conventional plastics, its biotechnological production is quite expensive. As carbon substrates contribute greatly to P[3HB] production cost, the utilization of a cheaper carbon substrate and less demanding micro-organisms should decrease its cost. In the present study a 23 factorial experimental design was applied, aiming to evaluate the effects of using hydrolysed corn starch (HCS) and soybean oil (SBO) as carbon substrates, and cheese whey (CW) supplementation in the mineral medium (MM) on the responses, cell dried weigh (DCW), percentage P[3HB] and mass P[3HB] by recombinant Escherichia coli strains JM101 and DH10B, containing the P[3HB] synthase genes from Cupriavidus necator (ex-Ralstonia eutropha). The analysis of effects indicated that the substrates and the supplement and their interactions had positive effect on CDW. Statistically generated equations showed that, at the highest concentrations of HCS, SO and CW, theoretically it should be possible to produce about 2 g L(1) DCW, accumulating 50% P[3HB], in both strains. To complement this study, the strain that presented the best results was cultivated in MM added to HCS, SBO and CW ( in best composition observed) and complex medium (CM) to compare the obtained P[3HB] in terms of physicochemical parameters. The obtained results showed that the P[3HB] production in MM (1.29 g L(-1)) was approximately 20% lower than in CM (1.63 g L(-1)); however, this difference can be compensated by the lower cost of the MM achieved by the use of cheap renewable carbon sources. Moreover, using differential scanning calorimetry and thermogravimetry analyses, it was observed that the polymer produced in MM was the one which presented physicochemical properties (Tg and Tf) that were more similar to those found in the literature for P[3HB].

Expression of soluble and functional full-length human matrix metalloproteinase-2 in Escherichia coli

Characterization of the matrix metalloproteinase-2 (MMP-2) substrates and understanding of its function remain difficult because up to date preparations containing minor amounts of other eukaryotic proteins that are co-purified with MMP-2 are still used. In this work, the expression of a soluble and functional full-length recombinant human MMP-2 (rhMMP-2) in the cytoplasm of Escherichia coli is reported, and the purification of this metalloproteinase is described. Culture of this bacterium at 18 degrees C culminated in maintenance of the soluble and functional rhMMP-2 in the soluble fraction of the E. coli lysate and its purification by affinity with gelatin-sepharose yielded approximately 0.12 mg/L of medium. Western Blotting and zymographic analysis revealed that the most abundant form was the 72-kDa MMP-2, but some gelatinolytic bands corresponding to proteins with lower molecular weight were also detected. The obtained rhMMP-2 was demonstrated to be functional in a gelatinolytic fluorimetric assay, suggesting that the purified rhMMP-2 was correctly folded. The method described here involves fewer steps, is less expensive, and is less prone to contamination with other proteinases and MMP inhibitors as compared to expression of rhMMP-2 in eukaryotic tissue culture. This protocol will facilitate the use of the full-length rhMMP-2 expressed in bacteria and will certainly help researchers to acquire new knowledge about the substrates and biological activities of this important proteinase. (C) 2011 Elsevier B.V. All rights reserved.

Milk hygienic practices and occurrence of Staphylococcus aureus and Escherichia coli O157:H7 in small-scale dairy farms in Sao Paulo, Brazil

The objective of this study was to assess the relationship between somatic cell counts (SCC), the use of different milking practices, and the occurrence of Staphylococcus aureus and Escherichia coli O157:H7 in 42 small-scale dairy farms located in the state of Sao Paulo, Brazil. S. aureus and E. coli O157:H7 were isolated in the milk from dairy cows with low (< 200,000 cells/ml) and high SCC (>200,000 cells/ml), although no effect of SCC (p > 0.05) was observed on the incidence of the bacteria in raw milk. The use of disposable gloves during milking reduced S. aureus counts in milk (p < 0.05), but did not affect the occurrence of E. coli O157:H7. The other milking practices evaluated (closed milking system, use of pre- and post-dipping, mastitis diagnosis by strip cup test, and disinfection of teat cups) did not affect (p < 0.05) the occurrence of S. aureus or E. coli O157:H7 in raw milk. Results indicate the need for effective educational programs addressed to prevent the contamination of milk with S. aureus and E. coli O157:H7 in Brazilian small-scale dairy farms.

Expression of recombinant human mutant granulocyte colony stimulating factor (Nartograstim) in Escherichia coli

The human granulocyte colony stimulating factor (hG-CSF) plays an important role in hematopoietic cell proliferation/differentiation and has been widely used as a therapeutic agent for treating neutropenias. Nartograstim is a commercial G-CSF that presents amino acid changes in specific positions when compared to the wildtype form, which potentially increase its activity and stability. The aim of this work was to develop an expression system in Escherichia coli that leads to the production of large amounts of a recombinant hG-CSF (rhG-CSF) biosimilar to Nartograstim. The nucleotide sequence of hg-csf was codon-optimized for expression in E. coli. As a result, high yields of the recombinant protein were obtained with adequate purity, structural integrity and biological activity. This protein has also been successfully used for the production of specific polyclonal antibodies in mice, which could be used in the control of the expression and purification in an industrial production process of this recombinant protein. These results will allow the planning of large-scale production of this mutant version of hG-CSF (Nartograstim), as a potential new biosimilar in the market.

Extensive cross-talk and global regulators identified from an analysis of the integrated transcriptional and signaling network in Escherichia coli

To understand the regulatory dynamics of transcription factors (TFs) and their interplay with other cellular components we have integrated transcriptional, protein-protein and the allosteric or equivalent interactions which mediate the physiological activity of TFs in Escherichia coli. To study this integrated network we computed a set of network measurements followed by principal component analysis (PCA), investigated the correlations between network structure and dynamics, and carried out a procedure for motif detection. In particular, we show that outliers identified in the integrated network based on their network properties correspond to previously characterized global transcriptional regulators. Furthermore, outliers are highly and widely expressed across conditions, thus supporting their global nature in controlling many genes in the cell. Motifs revealed that TFs not only interact physically with each other but also obtain feedback from signals delivered by signaling proteins supporting the extensive cross-talk between different types of networks. Our analysis can lead to the development of a general framework for detecting and understanding global regulatory factors in regulatory networks and reinforces the importance of integrating multiple types of interactions in underpinning the interrelationships between them.

Selective Permeation and Organic Extraction of Recombinant Green Fluorescent Protein (gfpuv) from Escherichia coli

Background Transformed cells of Escherichia coli DH5-α with pGFPuv, induced by IPTG (isopropyl-β-d-thiogalactopyranoside), express the green fluorescent protein (gfpuv) during growth phases. E. coli subjected to the combination of selective permeation by freezing/thawing/sonication cycles followed by the three-phase partitioning extraction (TPP) method were compared to the direct application of TPP to the same culture of E. coli on releasing gfpuv from the over-expressing cells. Material and Methods Cultures (37°C/100 rpm/ 24 h; μ = 0.99 h-1 - 1.10 h-1) of transformed (pGFP) Escherichia coli DH5-α, expressing the green fluorescent protein (gfpuv, absorbance at 394 nm and emission at 509 nm) were sonicated in successive intervals of sonication (25 vibrations/pulse) to determine the maximum amount of gfpuv released from the cells. For selective permeation, the transformed previously frozen (-75°C) cells were subjected to three freeze/thaw (-20°C/ 0.83°C/min) cycles interlaid by sonication (3 pulses/ 6 seconds/ 25 vibrations). The intracellular permeate with gfpuv in extraction buffer (TE) solution (25 mM Tris-HCl, pH 8.0, 1 mM β-mercaptoethanol β-ME, 0.1 mM PMSF) was subjected to the three-phase partitioning (TPP) method with t-butanol and 1.6 M ammonium sulfate. Sonication efficiency was verified on the application to the cells previously treated by the TPP method. The intra-cell releases were mixed and eluted through methyl HIC column with a buffer solution (10 mM Tris-HCl, 10 mM EDTA, pH 8.0). Results The sonication maximum released amount obtained from the cells was 327.67 μg gfpuv/mL (20.73 μg gfpuv/mg total proteins – BSA), after 9 min of treatment. Through the selective permeation by three repeated freezing/thawing/sonication cycles applied to the cells, a close content of 241.19 μg gfpuv/mL (29.74 μg gfpuv/mg BSA) was obtained. The specific mass range of gfpuv released from the same cultures, by the three-phase partitioning (TPP) method, in relation to total proteins, was higher, between 107.28 μg/mg and 135.10 μg/mg. Conclusions The selective permeation of gfpuv by freezing/thawing/sonication followed by TPP separation method was equivalent to the amount of gfpuv extracted from the cells directly by TPP; although selective permeation extracts showed better elution through the HIC column.

Expression of human protein S100A7 (psoriasin), preparation of antibody and application to human larynx squamous cell carcinoma

Background Up-regulation of S100A7 (Psoriasin), a small calcium-binding protein, is associated with the development of several types of carcinomas, but its function and possibility to serve as a diagnostic or prognostic marker have not been fully defined. In order to prepare antibodies to the protein for immunohistochemical studies we produced the recombinant S100A7 protein in E. coli. mRNA extracted from human tracheal tumor tissue which was amplified by RT-PCR to provide the region coding for the S100A7 gene. The amplified fragment was cloned in the vector pCR2.1-TOPO and sub-cloned in the expression vector pAE. The protein rS100A7 (His-tag) was expressed in E. coli BL21::DE3, purified by affinity chromatography on an Ni-NTA column, recovered in the 2.0 to 3.5 mg/mL range in culture medium, and used to produce a rabbit polyclonal antibody anti-rS100A7 protein. The profile of this polyclonal antibody was evaluated in a tissue microarray. Results The rS100A7 (His-tag) protein was homogeneous by SDS-PAGE and mass spectrometry and was used to produce an anti-recombinant S100A7 (His-tag) rabbit serum (polyclonal antibody anti-rS100A7). The molecular weight of rS100A7 (His-tag) protein determined by linear MALDI-TOF-MS was 12,655.91 Da. The theoretical mass calculated for the nonapeptide attached to the amino terminus is 12,653.26 Da (delta 2.65 Da). Immunostaining with the polyclonal anti-rS100A7 protein generated showed reactivity with little or no background staining in head and neck squamous cell carcinoma cells, detecting S100A7 both in nucleus and cytoplasm. Lower levels of S100A7 were detected in non-neoplastic tissue. Conclusions The polyclonal anti-rS100A7 antibody generated here yielded a good signal-to-noise contrast and should be useful for immunohistochemical detection of S100A7 protein. Its potential use for other epithelial lesions besides human larynx squamous cell carcinoma and non-neoplastic larynx should be explored in future.