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Cell-free protein synthesis is a rapid and efficient method for the production of recombinant proteins. Usage of prokaryotic cell-free extracts often leads to non-functional proteins. Eukaryotic counterparts such as wheat germ extract (WGE) and rabbit reticulocyte lysate (RLL) may improve solubility and promote the correct folding of eukaryotic multi-domain proteins that are difficult to express in bacteria. However, the preparation of WGEs is complex and time-consuming, whereas RLLs suffer from low yields. Here we report the development of a novel cell-free system based on tobacco Bright Yellow 2 (BY-2) cells harvested in the exponential growth phase. The highly-productive BY-2 lysate (BYL) can be prepared quickly within 4–5 h, compared to 4–5 d for WGE. The efficiency of the BYL was tested using three model proteins: enhanced yellow fluorescent protein (eYFP) and two versions of luciferase. The added mRNA was optimized by testing different 5’ and 3’ untranslated regions (UTRs). The protein yield in batch and dialysis reactions using BYL was much higher than that of a commercial Promega WGE preparation, achieving a maximum yield of 80 μg/mL of eYFP and 100 μg/mL of luciferase, compared to only 45 μg/mL of eYFP and 35 μg/mL of luciferase in WGEs. In dialysis reactions, the BYL yielded about 400 μg/mL eYFP, representing up to 50% more of the target protein than the Promega WGE, and equivalent to the amount using 5Prime WGE system. Due to the high yield and the short preparation time the BYL represents a remarkable improvement over current eukaryotic cell-free systems.

Microbial polysaccharides have been reported to possess remarkable bioactivities. Physarum polycephalum is a species of slime mold for which the microplasmodia are capable of rapid growth and can produce a significant amount of cell wall-less biomass. There has been a limited understanding of the polysaccharides produced by microplasmodia of slime molds, including P. polycephalum. Thus, the primary objectives of this research were first to chemically characterize the exopolysaccharides (EPS) and intracellular polysaccharides (IPS) of P. polycephalum microplasmodia and then to evaluate their cytotoxicity against several cancer cell lines. The yields of the crude EPS (4.43 ± 0.44 g/l) and partially purified (deproteinated) EPS (2.95 ± 0.85 g/l) were comparable (p > 0.05) with the respective crude IPS (3.46 ± 0.36 g/l) and partially purified IPS (2.45 ± 0.36 g/l). The average molecular weight of the EPS and IPS were 14,762 kDa and 1788 kDa. The major monomer of the EPS was galactose (80.22%), while that of the IPS was glucose (84.46%). Both crude and purified IPS samples showed significantly higher cytotoxicity toward Hela cells, especially the purified sample and none of the IPSs inhibited normal cells. Only 38.42 ± 2.84% Hela cells remained viable when treated with the partially purified IPS (1 mg/ml). However, although only 34.76 ± 6.58% MCF-7 cells were viable when exposed to the crude IPS, but the partially purified IPS displayed non-toxicity to MCF-7 cells. This suggested that the cytotoxicity toward MCF-7 would come from some component associated with the crude IPS sample (e.g. proteins, peptides or ion metals) and the purification process would have either completely removed or reduced amount of that component. Cell cycle analysis by flow cytometry suggested that the mechanism of the toxicity of the crude IPS toward MCF-7 and the partially purified IPS toward Hela cells was due to apoptosis. The EPS and IPS of P. polycephalum microplasmodia had different chemical properties including carbohydrate, protein and total sulfate group contents, monosaccharide composition and molecular weights, which led to different cytotoxicity activities. The crude and partially purified IPSs would be potential materials for further study relating to cancer treatment.

Despite the proven relevance of Pseudomonas fluorescens as a spoilage microorganism in milk, fresh meats and refrigerated food products and the recognized potential of bacteriophages as sanitation agents, so far no phages specific for P. fluorescens isolates from dairy industry have been closely characterized in view of their lytic efficiency. Here we describe the isolation and characterization of a lytic phage capable to infect a variety of P. fluorescens strains isolated from Portuguese and United States dairy industries. Several phages were isolated which showed a different host spectrum and efficiency of lysis. One of the phages, phage ϕIBB-PF7A, was studied in detail due to its efficient lysis of a wide spectrum of P. fluorescens strains and ribotypes. Phage ϕIBB-PF7A with a head diameter of about 63 nm and a tail size of about 13 × 8 nm belongs morphologically to the Podoviridae family and resembles a typical T7-like phage, as analyzed by transmission electron microscopy (TEM). The phage growth cycle with a detected latent period of 15 min, an eclipse period of 10 min, a burst size of 153 plaque forming units per infected cell, its genome size of approximately 42 kbp, and the size and N-terminal sequence of one of the protein bands, which gave similarity to the major capsid protein 10A, are consistent with this classification. The isolated T7-like phage, phage ϕIBB-PF7A, is fast and efficient in lysing different P. fluorescens strains and may be a good candidate to be used as a sanitation agent to control the prevalence of spoilage causing P. fluorescens strains in dairy and food related environments.

Spider silk is a tear-resistant and elastic biopolymer that has outstanding mechanical properties. Additionally, exiguous immunogenicity is anticipated for spider silks. Therefore, spider silk represents a potential ideal biomaterial for medical applications. All known spider silk proteins, so-called spidroins, reveal a composite nature of silk-specific units, allowing the recombinant production of individual and combined segments. In this report, a miniaturized spidroin gene, named VSO1 that contains repetitive motifs of MaSp1 has been synthesized and combined to form multimers of distinct lengths, which were heterologously expressed as elastin-like peptide (ELP) fusion proteins in tobacco. The elastic penetration moduli of layered proteins were analyzed for different spidroin-based biopolymers. Moreover, we present the first immunological analysis of synthetic spidroin-based biopolymers. Characterization of the binding behavior of the sera after immunization by competitive ELISA suggested that the humoral immune response is mainly directed against the fusion partner ELP. In addition, cytocompatibility studies with murine embryonic fibroblasts indicated that recombinant spidroin-based biopolymers, in solution or as coated proteins, are well tolerated. The results show that spidroin-based biopolymers can induce humoral immune responses that are dependent on the fusion partner and the overall protein structure. Furthermore, cytocompatibility assays gave no indication of spidroin-derived cytotoxicity, suggesting that recombinant produced biopolymers composed of spider silk-like repetitive elements are suitable for biomedical applications.

Circulating microRNAs are undergoing exploratory use as safety biomarkers in drug development. Reverse transcription quantitative polymerase chain reaction (RT-qPCR) is one common approach used to quantitate levels of microRNAs in samples that includes the use of a standard curve of calibrators fit to a regression model. Guidelines are needed for setting assay quantitation thresholds that are appropriate for this method and to biomarker pre-validation. In this report, we develop two workflows for determining a lower limit of quantitation (LLOQ) for RT-qPCR assays of microRNAs in exploratory studies. One workflow is based on an error threshold calculated by a logistic model of the calibration curve data. The second workflow is based on a threshold set by the sample blank, which is the no template control for RT-qPCR. The two workflows are used to set lower thresholds of reportable microRNA levels for an example dataset in which miR-208a levels in biofluids are quantitated in a cardiac injury model. LLOQ thresholds set by either workflow are effective in filtering out microRNA values with large uncertainty estimates. Two workflows for LLOQ determinations are presented in this report that provide methods that are easy to implement in investigational studies of microRNA safety biomarkers and offer choices in levels of conservatism in setting lower limits of acceptable values that facilitate interpretation of results.

Abstract Background Basidiomycete high-redox potential laccases (HRPLs) working in human physiological fluids (pH 7.4, 150 mM NaCl) arise great interest in the engineering of 3D-nanobiodevices for biomedical uses. In two previous reports, we described the directed evolution of a HRPL from basidiomycete PM1 strain CECT 2971: i) to be expressed in an active, soluble and stable form in Saccharomyces cerevisiae, and ii) to be active in human blood. In spite of the fact that S. cerevisiae is suited for the directed evolution of HRPLs, the secretion levels obtained in this host are not high enough for further research and exploitation. Thus, the search for an alternative host to over-express the evolved laccases is mandatory. Results A blood-active laccase (ChU-B mutant) fused to the native/evolved α-factor prepro-leader was cloned under the control of two different promoters (PAOX1 and PGAP) and expressed in Pichia pastoris. The most active construct, which contained the PAOX1 and the evolved prepro-leader, was fermented in a 42-L fed-batch bioreactor yielding production levels of 43 mg/L. The recombinant laccase was purified to homogeneity and thoroughly characterized. As happened in S. cerevisiae, the laccase produced by P. pastoris presented an extra N-terminal extension (ETEAEF) generated by an alternative processing of the α-factor pro-leader at the Golgi compartment. The laccase mutant secreted by P. pastoris showed the same improved properties acquired after several cycles of directed evolution in S. cerevisiae for blood-tolerance: a characteristic pH-activity profile shifted to the neutral-basic range and a greatly increased resistance against inhibition by halides. Slight biochemical differences between both expression systems were found in glycosylation, thermostability and turnover numbers. Conclusions The tandem-yeast system based on S. cerevisiae to perform directed evolution and P. pastoris to over-express the evolved laccases constitutes a promising approach for the in vitro evolution and production of these enzymes towards different biocatalytic and bioelectrochemical applications.

Abstract Background The use of lactic acid bacteria as vehicles to delivery antigens to immunize animals is a promising issue. When genetically modified, these bacteria can induce a specific local and systemic immune response against selected pathogens. Gastric acid and bile salts tolerance, production of antagonistic substances against pathogenic microorganisms, and adhesive ability to gut epithelium are other important characteristics that make these bacteria useful for oral immunization. Results Bacteria isolated on de Man, Rogosa and Sharpe medium (MRS) from different gastrointestinal portions of broiler chicks were evaluated for their resistance to artificial gastric acid and bile salts, production of hydrogen peroxide, and cell surface hydrophobicity. Thirty-eight isolates were first typed at species level by PCR amplification of 16S-23S rRNA intergenic spacers using universal primers that anneal within 16S and 23S genes, followed by restriction digestion analyses of PCR amplicons (PCR-ARDRA). An expression cassette was assembled onto the pCR2.1-Topo vector by cloning the promoter, leader peptide, cell wall anchor and terminator sequences derived from the laminin binding S-layer protein gene of L. crispatus strain F5.7 (lbs gene). A sequence encoding the green fluorescent protein (GFP) was inserted as reporter gene, and an erythromycin resistance gene was added as selective marker. All constructs were able to express GFP in the cloning host E. coli XL1-Blue and different Lactobacillus strains as verified by FACS and laser scanning confocal microscopy. Conclusion Lactobacillus isolated from gastrointestinal tract of broiler chickens and selected for probiotic characteristics can be genetically modified by introducing an expression cassette into the lbs locus. The transformed bacteria expressed on its cell wall surface different fluorescent proteins used as reporters of promoter function. It is possible then that similar bacterial model expressing pathogen antigens can be used as live oral vaccines to immunize broilers against infectious diseases.