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Sample records for microorganisms producing recombinant

  1. Recombinant microorganisms for increased production of organic acids

    DOEpatents

    Yi, Jian; Kleff, Susanne; Guettler, Michael V

    2013-04-30

    Disclosed are recombinant microorganisms for producing organic acids. The recombinant microorganisms express a polypeptide that has the enzymatic activity of an enzyme that is utilized in the pentose phosphate cycle. The recombinant microorganism may include recombinant Actinobacillus succinogenes that has been transformed to express a Zwischenferment (Zwf) gene. The recombinant microorganisms may be useful in fermentation processes for producing organic acids such as succinic acid and lactic acid. Also disclosed are novel plasmids that are useful for transforming microorganisms to produce recombinant microorganisms that express enzymes such as Zwf.

  2. Recombinant microorganisms for increased production of organic acids

    DOEpatents

    Yi, Jian [East Lansing, MI; Kleff, Susanne [East Lansing, MI; Guettler, Michael V [Holt, MI

    2012-02-21

    Disclosed are recombinant microorganisms for producing organic acids. The recombinant microorganisms express a polypeptide that has the enzymatic activity of an enzyme that is utilized in the pentose phosphate cycle. The recombinant microorganism may include recombinant Actinobacillus succinogenes that has been transformed to express a Zwischenferment (Zwf) gene. The recombinant microorganisms may be useful in fermentation processes for producing organic acids such as succinic acid and lactic acid. Also disclosed are novel plasmids that are useful for transforming microorganisms to produce recombinant microorganisms that express enzymes such as Zwf.

  3. Biofuel production by recombinant microorganisms

    DOEpatents

    Liao, James C.; Atsumi, Shota; Cann, Anthony F.

    2017-07-04

    Provided herein are metabolically-modified microorganisms useful for producing biofuels. More specifically, provided herein are methods of producing high alcohols including isobutanol, 1-butanol, 1-propanol, 2-methyl-1-butanol, 3-methyl-1-butanol and 2-phenylethanol from a suitable substrate.

  4. Microorganisms for producing organic acids

    DOEpatents

    Pfleger, Brian Frederick; Begemann, Matthew Brett

    2014-09-30

    Organic acid-producing microorganisms and methods of using same. The organic acid-producing microorganisms comprise modifications that reduce or ablate AcsA activity or AcsA homolog activity. The modifications increase tolerance of the microorganisms to such organic acids as 3-hydroxypropionic acid, acrylic acid, propionic acid, lactic acid, and others. Further modifications to the microorganisms increase production of such organic acids as 3-hydroxypropionic acid, lactate, and others. Methods of producing such organic acids as 3-hydroxypropionic acid, lactate, and others with the modified microorganisms are provided. Methods of using acsA or homologs thereof as counter-selectable markers are also provided.

  5. Microorganisms for producing organic acids

    SciTech Connect

    Pfleger, Brian Frederick; Begemann, Matthew Brett

    2014-09-30

    Organic acid-producing microorganisms and methods of using same. The organic acid-producing microorganisms comprise modifications that reduce or ablate AcsA activity or AcsA homolog activity. The modifications increase tolerance of the microorganisms to such organic acids as 3-hydroxypropionic acid, acrylic acid, propionic acid, lactic acid, and others. Further modifications to the microorganisms increase production of such organic acids as 3-hydroxypropionic acid, lactate, and others. Methods of producing such organic acids as 3-hydroxypropionic acid, lactate, and others with the modified microorganisms are provided. Methods of using acsA or homologs thereof as counter-selectable markers are also provided.

  6. Cellulase producing microorganism ATCC 55702

    DOEpatents

    Dees, H.C.

    1997-12-30

    Bacteria which produce large amounts of cellulase--containing cell-free fermentate have been identified. The original bacterium (ATCC 55703) was genetically altered using nitrosoguanidine (MNNG) treatment to produce the enhanced cellulase producing bacterium (ATCC 55702), which was identified through replicate plating. ATCC 55702 has improved characteristics and qualifies for the degradation of cellulosic waste materials for fuel production, food processing, textile processing, and other industrial applications. ATCC 55702 is an improved bacterial host for genetic manipulations using recombinant DNA techniques, and is less likely to destroy genetic manipulations using standard mutagenesis techniques. 5 figs.

  7. Cellulase producing microorganism ATCC 55702

    DOEpatents

    Dees, H. Craig

    1997-01-01

    Bacteria which produce large amounts of cellulase--containing cell-free fermentate have been identified. The original bacterium (ATCC 55703) was genetically altered using nitrosoguanidine (MNNG) treatment to produce the enhanced cellulase producing bacterium (ATCC 55702), which was identified through replicate plating. ATCC 55702 has improved characteristics and qualifies for the degradation of cellulosic waste materials for fuel production, food processing, textile processing, and other industrial applications. ATCC 55702 is an improved bacterial host for genetic manipulations using recombinant DNA techniques, and is less likely to destroy genetic manipulations using standard mutagenesis techniques.

  8. Engineered microorganisms capable of producing target compounds under anaerobic conditions

    DOEpatents

    Buelter, Thomas [Denver, CO; Meinhold, Peter [Denver, CO; Feldman, Reid M. Renny [San Francisco, CA; Hawkins, Andrew C [Parker, CO; Urano, Jun [Irvine, CA; Bastian, Sabine [Pasadena, CA; Arnold, Frances [La Canada, CA

    2012-01-17

    The present invention is generally provides recombinant microorganisms comprising engineered metabolic pathways capable of producing C3-C5 alcohols under aerobic and anaerobic conditions. The invention further provides ketol-acid reductoisomerase enzymes which have been mutated or modified to increase their NADH-dependent activity or to switch the cofactor preference from NADPH to NADH and are expressed in the modified microorganisms. In addition, the invention provides isobutyraldehyde dehydrogenase enzymes expressed in modified microorganisms. Also provided are methods of producing beneficial metabolites under aerobic and anaerobic conditions by contacting a suitable substrate with the modified microorganisms of the present invention.

  9. Biocorrosion produced by Thiobacillus-like microorganisms.

    PubMed

    López, A I; Marín, I; Amils, R

    1994-01-01

    Biocorrosion can be produced by many different microorganisms through diverse mechanisms. The biocorrosion produced by acidophilic microorganisms of the genus Thiobacillus is based on the production of sulfuric acid and ferric ion from pyrites or related mineral structures, as a result of the chemolithotrophic metabolism of these microorganisms. The products of this aerobic respiration are also powerful oxidant elements, which can produce chemical oxidations of other metallic structures. The Tinto River, a very unusual extremophilic habitat (pH around 2, and high concentration of ferric ion), product of the growth of strict chemolithotrophic microorganisms, is discussed as a model case.

  10. Multiple microfermentor battery: a versatile tool for use with automated parallel cultures of microorganisms producing recombinant proteins and for optimization of cultivation protocols.

    PubMed

    Frachon, Emmanuel; Bondet, Vincent; Munier-Lehmann, Hélène; Bellalou, Jacques

    2006-08-01

    A multiple microfermentor battery was designed for high-throughput recombinant protein production in Escherichia coli. This novel system comprises eight aerated glass reactors with a working volume of 80 ml and a moving external optical sensor for measuring optical densities at 600 nm (OD600) ranging from 0.05 to 100 online. Each reactor can be fitted with miniature probes to monitor temperature, dissolved oxygen (DO), and pH. Independent temperature regulation for each vessel is obtained with heating/cooling Peltier devices. Data from pH, DO, and turbidity sensors are collected on a FieldPoint (National Instruments) I/O interface and are processed and recorded by a LabVIEW program on a personal computer, which enables feedback control of the culture parameters. A high-density medium formulation was designed, which enabled us to grow E. coli to OD600 up to 100 in batch cultures with oxygen-enriched aeration. Accordingly, the biomass and the amount of recombinant protein produced in a 70-ml culture were at least equivalent to the biomass and the amount of recombinant protein obtained in a Fernbach flask with 1 liter of conventional medium. Thus, the microfermentor battery appears to be well suited for automated parallel cultures and process optimization, such as that needed for structural genomics projects.

  11. Multiple Microfermentor Battery: a Versatile Tool for Use with Automated Parallel Cultures of Microorganisms Producing Recombinant Proteins and for Optimization of Cultivation Protocols

    PubMed Central

    Frachon, Emmanuel; Bondet, Vincent; Munier-Lehmann, Hélène; Bellalou, Jacques

    2006-01-01

    A multiple microfermentor battery was designed for high-throughput recombinant protein production in Escherichia coli. This novel system comprises eight aerated glass reactors with a working volume of 80 ml and a moving external optical sensor for measuring optical densities at 600 nm (OD600) ranging from 0.05 to 100 online. Each reactor can be fitted with miniature probes to monitor temperature, dissolved oxygen (DO), and pH. Independent temperature regulation for each vessel is obtained with heating/cooling Peltier devices. Data from pH, DO, and turbidity sensors are collected on a FieldPoint (National Instruments) I/O interface and are processed and recorded by a LabVIEW program on a personal computer, which enables feedback control of the culture parameters. A high-density medium formulation was designed, which enabled us to grow E. coli to OD600 up to 100 in batch cultures with oxygen-enriched aeration. Accordingly, the biomass and the amount of recombinant protein produced in a 70-ml culture were at least equivalent to the biomass and the amount of recombinant protein obtained in a Fernbach flask with 1 liter of conventional medium. Thus, the microfermentor battery appears to be well suited for automated parallel cultures and process optimization, such as that needed for structural genomics projects. PMID:16885269

  12. Food-processing enzymes from recombinant microorganisms--a review.

    PubMed

    Olempska-Beer, Zofia S; Merker, Robert I; Ditto, Mary D; DiNovi, Michael J

    2006-07-01

    Enzymes are commonly used in food processing and in the production of food ingredients. Enzymes traditionally isolated from culturable microorganisms, plants, and mammalian tissues are often not well-adapted to the conditions used in modern food production methods. The use of recombinant DNA technology has made it possible to manufacture novel enzymes suitable for specific food-processing conditions. Such enzymes may be discovered by screening microorganisms sampled from diverse environments or developed by modification of known enzymes using modern methods of protein engineering or molecular evolution. As a result, several important food-processing enzymes such as amylases and lipases with properties tailored to particular food applications have become available. Another important achievement is improvement of microbial production strains. For example, several microbial strains recently developed for enzyme production have been engineered to increase enzyme yield by deleting native genes encoding extracellular proteases. Moreover, certain fungal production strains have been modified to reduce or eliminate their potential for production of toxic secondary metabolites. In this article, we discuss the safety of microorganisms used as hosts for enzyme-encoding genes, the construction of recombinant production strains, and methods of improving enzyme properties. We also briefly describe the manufacture and safety assessment of enzyme preparations and summarize options for submitting information on enzyme preparations to the US Food and Drug Administration.

  13. Clostridiumm ljungdahlii, an anaerobic ethanol and acetate producing microorganism

    DOEpatents

    Gaddy, J.L.; Clausen, E.C.

    1992-12-22

    A newly discovered microorganism was isolated in a biologically pure culture and designated Clostridium ljungdahlii, having the identifying characteristics of ATCC No. 49587. Cultured in an aqueous nutrient medium under anaerobic conditions, this microorganism is capable of producing ethanol and acetate from CO and H[sub 2]O and/or CO[sub 2] and H[sub 2] in synthesis gas. Under optimal growth conditions, the microorganism produces acetate in preference to ethanol. Conversely, under non-growth conditions, ethanol production is favored over acetate. 3 figs.

  14. Clostridiumm ljungdahlii, an anaerobic ethanol and acetate producing microorganism

    DOEpatents

    Gaddy, James L.; Clausen, Edgar C.

    1992-01-01

    A newly discovered microorganism was isolated in a biologically pure culture and designated Clostridium ljungdahlii, having the identifying characteristics of ATCC No. 49587. Cultured in an aqueous nutrient medium under anaerobic conditions, this microorganism is capable of producing ethanol and acetate from CO and H.sub.2 O and/or CO.sub.2 and H.sub.2 in synthesis gas. Under optimal growth conditions, the microorganism produces acetate in preference to ethanol. Conversely, under non-growth conditions, ethanol production is favored over acetate.

  15. Biosurfactants Produced by Marine Microorganisms with Therapeutic Applications.

    PubMed

    Gudiña, Eduardo J; Teixeira, José A; Rodrigues, Lígia R

    2016-02-18

    Marine microorganisms possess unique metabolic and physiological features and are an important source of new biomolecules, such as biosurfactants. Some of these surface-active compounds synthesized by marine microorganisms exhibit antimicrobial, anti-adhesive and anti-biofilm activity against a broad spectrum of human pathogens (including multi-drug resistant pathogens), and could be used instead of existing drugs to treat infections caused by them. In other cases, these biosurfactants show anti-cancer activity, which could be envisaged as an alternative to conventional therapies. However, marine biosurfactants have not been widely explored, mainly due to the difficulties associated with the isolation and growth of their producing microorganisms. Culture-independent techniques (metagenomics) constitute a promising approach to study the genetic resources of otherwise inaccessible marine microorganisms without the requirement of culturing them, and can contribute to the discovery of novel biosurfactants with significant biological activities. This paper reviews the most relevant biosurfactants produced by marine microorganisms with potential therapeutic applications and discusses future perspectives and opportunities to discover novel molecules from marine environments.

  16. Biosurfactants Produced by Marine Microorganisms with Therapeutic Applications

    PubMed Central

    Gudiña, Eduardo J.; Teixeira, José A.; Rodrigues, Lígia R.

    2016-01-01

    Marine microorganisms possess unique metabolic and physiological features and are an important source of new biomolecules, such as biosurfactants. Some of these surface-active compounds synthesized by marine microorganisms exhibit antimicrobial, anti-adhesive and anti-biofilm activity against a broad spectrum of human pathogens (including multi-drug resistant pathogens), and could be used instead of existing drugs to treat infections caused by them. In other cases, these biosurfactants show anti-cancer activity, which could be envisaged as an alternative to conventional therapies. However, marine biosurfactants have not been widely explored, mainly due to the difficulties associated with the isolation and growth of their producing microorganisms. Culture-independent techniques (metagenomics) constitute a promising approach to study the genetic resources of otherwise inaccessible marine microorganisms without the requirement of culturing them, and can contribute to the discovery of novel biosurfactants with significant biological activities. This paper reviews the most relevant biosurfactants produced by marine microorganisms with potential therapeutic applications and discusses future perspectives and opportunities to discover novel molecules from marine environments. PMID:26901207

  17. Engineering biofuel tolerance in non-native producing microorganisms.

    PubMed

    Jin, Hu; Chen, Lei; Wang, Jiangxin; Zhang, Weiwen

    2014-01-01

    Large-scale production of renewable biofuels through microbiological processes has drawn significant attention in recent years, mostly due to the increasing concerns on the petroleum fuel shortages and the environmental consequences of the over-utilization of petroleum-based fuels. In addition to native biofuel-producing microbes that have been employed for biofuel production for decades, recent advances in metabolic engineering and synthetic biology have made it possible to produce biofuels in several non-native biofuel-producing microorganisms. Compared to native producers, these non-native systems carry the advantages of fast growth, simple nutrient requirements, readiness for genetic modifications, and even the capability to assimilate CO2 and solar energy, making them competitive alternative systems to further decrease the biofuel production cost. However, the tolerance of these non-native microorganisms to toxic biofuels is naturally low, which has restricted the potentials of their application for high-efficiency biofuel production. To address the issues, researches have been recently conducted to explore the biofuel tolerance mechanisms and to construct robust high-tolerance strains for non-native biofuel-producing microorganisms. In this review, we critically summarize the recent progress in this area, focusing on three popular non-native biofuel-producing systems, i.e. Escherichia coli, Lactobacillus and photosynthetic cyanobacteria.

  18. Medicinally important secondary metabolites in recombinant microorganisms or plants: progress in alkaloid biosynthesis.

    PubMed

    Schäfer, Holger; Wink, Michael

    2009-12-01

    Plants produce a high diversity of natural products or secondary metabolites which are important for the communication of plants with other organisms. A prominent function is the protection against herbivores and/or microbial pathogens. Some natural products are also involved in defence against abiotic stress, e.g. UV-B exposure. Many of the secondary metabolites have interesting biological properties and quite a number are of medicinal importance. Because the production of the valuable natural products, such as the anticancer drugs paclitaxel, vinblastine or camptothecin in plants is a costly process, biotechnological alternatives to produce these alkaloids more economically become increasingly important. This review provides an overview of the state of art to produce alkaloids in recombinant microorganisms, such as bacteria or yeast. Some progress has been made in metabolic engineering usually employing a single recombinant alkaloid gene. More importantly, for benzylisoquinoline, monoterpene indole and diterpene alkaloids (taxanes) as well as some terpenoids and phenolics the proof of concept for production of complex alkaloids in recombinant Escherichia coli and yeast has already been achieved. In a long-term perspective, it will probably be possible to generate gene cassettes for complete pathways, which could then be used for production of valuable natural products in bioreactors or for metabolic engineering of crop plants. This will improve their resistance against herbivores and/or microbial pathogens.

  19. Cultivating Insect Cells To Produce Recombinant Proteins

    NASA Technical Reports Server (NTRS)

    Spaulding, Glenn; Goodwin, Thomas; Prewett, Tacey; Andrews, Angela; Francis, Karen; O'Connor, Kim

    1996-01-01

    Method of producing recombinant proteins involves growth of insect cells in nutrient solution in cylindrical bioreactor rotating about cylindrical axis, oriented horizontally and infecting cells with viruses into which genes of selected type cloned. Genes in question those encoding production of desired proteins. Horizontal rotating bioreactor preferred for use in method, denoted by acronym "HARV", described in "High-Aspect-Ratio Rotating Cell-Culture Vessel" (MSC-21662).

  20. Cultivating Insect Cells To Produce Recombinant Proteins

    NASA Technical Reports Server (NTRS)

    Spaulding, Glenn; Goodwin, Thomas; Prewett, Tacey; Andrews, Angela; Francis, Karen; O'Connor, Kim

    1996-01-01

    Method of producing recombinant proteins involves growth of insect cells in nutrient solution in cylindrical bioreactor rotating about cylindrical axis, oriented horizontally and infecting cells with viruses into which genes of selected type cloned. Genes in question those encoding production of desired proteins. Horizontal rotating bioreactor preferred for use in method, denoted by acronym "HARV", described in "High-Aspect-Ratio Rotating Cell-Culture Vessel" (MSC-21662).

  1. Biofuel and chemical production by recombinant microorganisms via fermentation of proteinaceous biomass

    DOEpatents

    Liao, James C.; Cho, Kwang Myung; Yan, Yajun; Huo, Yixin

    2016-03-15

    Provided herein are metabolically modified microorganisms characterized by having an increased keto-acid flux when compared with the wild-type organism and comprising at least one polynucleotide encoding an enzyme that when expressed results in the production of a greater quantity of a chemical product when compared with the wild-type organism. The recombinant microorganisms are useful for producing a large number of chemical compositions from various nitrogen containing biomass compositions and other carbon sources. More specifically, provided herein are methods of producing alcohols, acetaldehyde, acetate, isobutyraldehyde, isobutyric acid, n-butyraldehyde, n-butyric acid, 2-methyl-1-butyraldehyde, 2-methyl-1-butyric acid, 3-methyl-1-butyraldehyde, 3-methyl-1-butyric acid, ammonia, ammonium, amino acids, 2,3-butanediol, 1,4-butanediol, 2-methyl-1,4-butanediol, 2-methyl-1,4-butanediamine, isobutene, itaconate, acetoin, acetone, isobutene, 1,5-diaminopentane, L-lactic acid, D-lactic acid, shikimic acid, mevalonate, polyhydroxybutyrate (PHB), isoprenoids, fatty acids, homoalanine, 4-aminobutyric acid (GABA), succinic acid, malic acid, citric acid, adipic acid, p-hydroxy-cinnamic acid, tetrahydrofuran, 3-methyl-tetrahydrofuran, gamma-butyrolactone, pyrrolidinone, n-methylpyrrolidone, aspartic acid, lysine, cadeverine, 2-ketoadipic acid, and/or S-adenosyl-methionine (SAM) from a suitable nitrogen rich biomass.

  2. Extracellular enzymes produced by microorganisms isolated from maritime Antarctica.

    PubMed

    Loperena, Lyliam; Soria, Verónica; Varela, Hermosinda; Lupo, Sandra; Bergalli, Alejandro; Guigou, Mairan; Pellegrino, Andrés; Bernardo, Angela; Calviño, Ana; Rivas, Federico; Batista, Silvia

    2012-05-01

    Antarctic environments can sustain a great diversity of well-adapted microorganisms known as psychrophiles or psychrotrophs. The potential of these microorganisms as a resource of enzymes able to maintain their activity and stability at low temperature for technological applications has stimulated interest in exploration and isolation of microbes from this extreme environment. Enzymes produced by these organisms have a considerable potential for technological applications because they are known to have higher enzymatic activities at lower temperatures than their mesophilic and thermophilic counterparts. A total of 518 Antarctic microorganisms, were isolated during Antarctic expeditions organized by the Instituto Antártico Uruguayo. Samples of particules suspended in air, ice, sea and freshwater, soil, sediment, bird and marine animal faeces, dead animals, algae, plants, rocks and microbial mats were collected from different sites in maritime Antarctica. We report enzymatic activities present in 161 microorganisms (120 bacteria, 31 yeasts and 10 filamentous fungi) isolated from these locations. Enzymatic performance was evaluated at 4 and 20°C. Most of yeasts and bacteria grew better at 20°C than at 4°C, however the opposite was observed with the fungi. Amylase, lipase and protease activities were frequently found in bacterial strains. Yeasts and fungal isolates typically exhibited lipase, celullase and gelatinase activities. Bacterial isolates with highest enzymatic activities were identified by 16S rDNA sequence analysis as Pseudomonas spp., Psychrobacter sp., Arthrobacter spp., Bacillus sp. and Carnobacterium sp. Yeasts and fungal strains, with multiple enzymatic activities, belonged to Cryptococcus victoriae, Trichosporon pullulans and Geomyces pannorum.

  3. [Tobacco--a producer of recombinant interleukins].

    PubMed

    Budzianowski, Jaromir

    2012-01-01

    Interleukins are cytokines of highly pleiotropic activity and they have high potential for application in the treatment of cancer and autoimmune diseases. Trials of recombinant interleukin production in plants relate almost exclusively to tobacco, where through the transformation of the nuclear genome (agroinfection) monomeric (IL-2, IL-4, IL-13, IL-18), homodimeric (IL-10) and single-chain heterodimeric (IL-12) interleukins have been obtained. The expression of IL-10 as a homodimer in the chloroplast genome could not be reached. Expression of the given interleukin was obtained in the leaves, cell culture and culture of hairy roots of tobacco. Interleukins obtained in tobacco showed similar in vitro biological activity as commercial ILs produced mostly in E. coli. Glycosylated IL-13 obtained in tobacco was much more resistant to proteolytic digestion than commercial non-glycosylated IL-13; therefore in the case of sufficiently large production it could be suitable for oral administration in the treatment of type I diabetes.

  4. [Biotests for mineral waters with natural and recombinant luminescent microorganisms].

    PubMed

    Deriabin, D G; Aleshina, E S

    2008-01-01

    We have developed methods of biotesting mineral waters involving use of natural or recombinant luminescent strains with elimination of the effect of salt concentration and pH. To overcome the adverse effect of high salt concentrations, disguising the action of chemical pollutants, a special method of mineral water sample preparation is proposed. In this method, the marine luminescent bacterium Photobacterium phosphoreum (Microbiosensor B17 677f) is used as a test object. Samples to be analyzed are supplemented with NaCl depending on their natural salt concentration to adjust it to 3 g/l. Another approach, more universal and efficient, involves pH adjustment in the samples to 7.5. This value is suitable for application of both Microbiosensor B17 677f and the recombinant Escherichia coli strain harboring the cloned lux operon of P. leiognathi (Ecolum 9). It has been shown that this treatment, retaining the natural luminescence level of the bacterial biosensors, allows bioluminescent detection of exogenous pollutants added to the samples, including benzene and Cr(VI).

  5. Recombinant Human Factor IX Produced from Transgenic Porcine Milk

    PubMed Central

    Lee, Meng-Hwan; Lin, Yin-Shen; Tu, Ching-Fu; Yen, Chon-Ho

    2014-01-01

    Production of biopharmaceuticals from transgenic animal milk is a cost-effective method for highly complex proteins that cannot be efficiently produced using conventional systems such as microorganisms or animal cells. Yields of recombinant human factor IX (rhFIX) produced from transgenic porcine milk under the control of the bovine α-lactalbumin promoter reached 0.25 mg/mL. The rhFIX protein was purified from transgenic porcine milk using a three-column purification scheme after a precipitation step to remove casein. The purified protein had high specific activity and a low ratio of the active form (FIXa). The purified rhFIX had 11.9 γ-carboxyglutamic acid (Gla) residues/mol protein, which approached full occupancy of the 12 potential sites in the Gla domain. The rhFIX was shown to have a higher isoelectric point and lower sialic acid content than plasma-derived FIX (pdFIX). The rhFIX had the same N-glycosylation sites and phosphorylation sites as pdFIX, but had a higher specific activity. These results suggest that rhFIX produced from porcine milk is physiologically active and they support the use of transgenic animals as bioreactors for industrial scale production in milk. PMID:24955355

  6. Recombinant human factor IX produced from transgenic porcine milk.

    PubMed

    Lee, Meng-Hwan; Lin, Yin-Shen; Tu, Ching-Fu; Yen, Chon-Ho

    2014-01-01

    Production of biopharmaceuticals from transgenic animal milk is a cost-effective method for highly complex proteins that cannot be efficiently produced using conventional systems such as microorganisms or animal cells. Yields of recombinant human factor IX (rhFIX) produced from transgenic porcine milk under the control of the bovine α-lactalbumin promoter reached 0.25 mg/mL. The rhFIX protein was purified from transgenic porcine milk using a three-column purification scheme after a precipitation step to remove casein. The purified protein had high specific activity and a low ratio of the active form (FIXa). The purified rhFIX had 11.9 γ-carboxyglutamic acid (Gla) residues/mol protein, which approached full occupancy of the 12 potential sites in the Gla domain. The rhFIX was shown to have a higher isoelectric point and lower sialic acid content than plasma-derived FIX (pdFIX). The rhFIX had the same N-glycosylation sites and phosphorylation sites as pdFIX, but had a higher specific activity. These results suggest that rhFIX produced from porcine milk is physiologically active and they support the use of transgenic animals as bioreactors for industrial scale production in milk.

  7. Methods for identifying lipoxygenase producing microorganisms on agar plates.

    PubMed

    Nyyssölä, Antti; Heshof, Ruud; Haarmann, Thomas; Eidner, Jasmin; Westerholm-Parvinen, Ann; Langfelder, Kim; Kruus, Kristiina; de Graaff, Leo; Buchert, Johanna

    2012-03-26

    Plate assays for lipoxygenase producing microorganisms on agar plates have been developed. Both potassium iodide-starch and indamine dye formation methods were effective for detecting soybean lipoxygenase activity on agar plates. A positive result was also achieved using the β-carotene bleaching method, but the sensitivity of this method was lower than the other two methods. The potassium iodide-starch and indamine dye formation methods were also applied for detecting lipoxygenase production by Trichoderma reesei and Pichia pastoris transformants expressing the lipoxygenase gene of the fungus Gaeumannomyces graminis. In both cases lipoxygenase production in the transformants could be identified. For detection of the G. graminis lipoxygenase produced by Aspergillus nidulans the potassium iodide-starch method was successful. When Escherichia coli was grown on agar and soybean lipoxygenase was applied on the culture lipoxygenase activity could clearly be detected by the indamine dye formation method. This suggests that the method has potential for screening of metagenomic libraries in E. coli for lipoxygenase activity.

  8. Bifunctional recombinant fusion enzyme between maltooligosyltrehalose synthase and maltooligosyltrehalose trehalohydrolase of thermophilic microorganism Metallosphaera hakonensis.

    PubMed

    Seo, Ju-Seok; An, Ju Hee; Cheong, Jong-Joo; Choi, Yang Do; Kim, Chung Ho

    2008-09-01

    MhMTS and MhMTH are trehalose (alpha-D-glucopyranosyl- [1,1]-alpha-D-glucopyranose) biosynthesis genes of the thermophilic microorganism Metallosphaera hakonensis, and encode a maltooligosyltrehalose synthase (MhMTS) and a maltooligosyltrehalose trehalohydrolase (MhMTH), respectively. In this study, the two genes were fused inframe in a recombinant DNA, and expressed in Escherichia coli to produce a bifunctional fusion enzyme, MhMTSH. Similar to the two-step reactions with MhMTS and MhMTH, the fusion enzyme catalyzed the sequential reactions on maltopentaose, maltotriosyltrehalose formation, and following hydrolysis, producing trehalose and maltotriose. Optimum conditions for the fusion enzyme-catalyzed trehalose synthesis were around 70 degrees and pH 5.0-6.0. The MhMTSH fusion enzyme exhibited a high degree of thermostability, retaining 80% of the activity when pre-incubated at 70 degrees for 48 h. The stability was gradually abolished by incubating the fusion enzyme at above 80 degrees . The MhMTSH fusion enzyme was active on various sizes of maltooligosaccharides, extending its substrate specificity to soluble starch, the most abundant natural source of trehalose production.

  9. Bioremediation of Industrial Waste Through Enzyme Producing Marine Microorganisms.

    PubMed

    Sivaperumal, P; Kamala, K; Rajaram, R

    2017-01-01

    Bioremediation process using microorganisms is a kind of nature-friendly and cost-effective clean green technology. Recently, biodegradation of industrial wastes using enzymes from marine microorganisms has been reported worldwide. The prospectus research activity in remediation area would contribute toward the development of advanced bioprocess technology. To minimize industrial wastes, marine enzymes could constitute a novel alternative in terms of waste treatment. Nowadays, the evidence on the mechanisms of bioremediation-related enzymes from marine microorganisms has been extensively studied. This review also will provide information about enzymes from various marine microorganisms and their complexity in the biodegradation of comprehensive range of industrial wastes.

  10. Lipase-producing microorganisms from a Kenyan alkaline soda lake.

    PubMed

    Vargas, Virginia A; Delgado, Osvaldo D; Hatti-Kaul, Rajni; Mattiasson, Bo

    2004-01-01

    Lipolytic enzyme production of 150 isolated strains from samples of Lake Bogoria (Kenya) was examined. Among these, fifteen isolates were selected on the basis of their lipolytic activities and subjected to morphological and 16S rRNA gene sequencing analyses for their identification. All the microorganisms have been selected under culture conditions with pH ranges between 7-10 and temperatures of 37-55 degrees C. Most of them showed optimal growth at 37 degrees C and tolerated salinity up to 10% (w/v). Ten of the isolates were Gram-negative, nine of which were closely related to the Pseudomonas cluster and one to the Halomonas cluster sharing high similarity profile with Halomonas desiderata. The remaining Gram-positive isolates were closely related to the Bacillus cluster, and were grouped with Bacillus halodurans, Bacillus alcalophilus and Bacillus licheniformis. Four members of the Bacillus cluster and the Halomonas sp. produced lipolytic activity under alkaline conditions, while others did so at neutral pH values.

  11. Six Siderophore-Producing Microorganisms Identified in Biological Soil Crusts

    NASA Astrophysics Data System (ADS)

    Noonan, K.; Anbar, A. D.; Garcia-Pichel, F.; Poret-peterson, A. T.; Hartnett, H. E.

    2011-12-01

    Biological soil crusts (BSCs) are diverse microbial communities that colonize soils in arid and semi-arid environments. Cyanobacteria in BSCs are pioneer organisms that increase ecosystem habitability by providing fixed carbon (C) and nitrogen (N) as well as by reducing water run-off and increasing infiltration. Photosynthesis and N fixation, in particular, require a variety of metals in large quantities, and yet, metals are predominantly insoluble in the environments where BSCs thrive. Therefore, BSC organisms must have efficient strategies for extracting metals from soil minerals. We hypothesized that BSC microbes, particularly the cyanobacteria, produce siderophores to serve their metal-acquisition needs. Siderophores are small organic compounds that bind Fe with high affinity and are produced by a variety of microorganisms, including cyanobacteria. Most siderophores bind Fe, primarily; however, some can also bind Mo, V, and Cu. Soil siderophores are released by microbes to increase the solubility of metals from minerals and to facilitate microbial uptake. Thus, siderophores serve as chemical weathering agents and provide a direct link between soil microbes and minerals. Studying siderophore production in BSCs provides insight into how BSCs tackle the challenge of acquiring insoluble metals, and may help conservationists determine useful fertilizers for BSC growth by facilitating metal acquisition. Biological soil crusts were collected near Moab, UT. Soil slurries were prepared in deionized water and transferred to modified BG-11 agar plates. The O-CAS agar plate assay was used to screen organisms for siderophore production. Siderophore producing microbes were isolated and identified by16S rRNA gene sequencing. Cultures were then grown in 3 L batch cultures under metal limitation, and siderophore presence was monitored using the traditional liquid CAS assay. After siderophore detection, cells were removed by centrifugation, organic compounds were separated using

  12. Method of producing a cellulase-containing cell-free fermentate produced from microorganism ATCC 55702

    DOEpatents

    Dees, H.C.

    1998-05-26

    Bacteria which produce large amounts of cellulose-containing cell-free fermentate have been identified. The original bacterium (ATCC 55703) was genetically altered using nitrosoguanidine (MNNG) treatment to produce the enhanced cellulase producing bacterium (ATCC 55702), which was identified through replicate plating. ATCC 55702 has improved characteristics and qualities for the degradation of cellulosic waste materials for fuel production, food processing, textile processing, and other industrial applications. ATCC 55702 is an improved bacterial host for genetic manipulations using recombinant DNA techniques, and is less likely to destroy genetic manipulations using standard mutagenesis techniques. 5 figs.

  13. Method of producing a cellulase-containing cell-free fermentate produced from microorganism ATCC 55702

    DOEpatents

    Dees, H. Craig

    1998-01-01

    Bacteria which produce large amounts of cellulose-containing cell-free fermentate have been identified. The original bacterium (ATCC 55703) was genetically altered using nitrosoguanidine (MNNG) treatment to produce the enhanced cellulase producing bacterium (ATCC 55702), which was identified through replicate plating. ATCC 55702 has improved characteristics and qualities for the degradation of cellulosic waste materials for fuel production, food processing, textile processing, and other industrial applications. ATCC 55702 is an improved bacterial host for genetic manipulations using recombinant DNA techniques, and is less likely to destroy genetic manipulations using standard mutagenesis techniques.

  14. Alum affects ammonia-producing microorganisms in poultry litter

    USDA-ARS?s Scientific Manuscript database

    Scientists at the USDA-ARS in Bowling Green, KY and in Fayetteville, AR are working to uncover the microbiology of ammonia production in poultry litter. Poultry litter is a valuable nutrient source for plants and microorganisms that contains high levels of protein, nitrogen, and other minerals. Howe...

  15. Advancing oleaginous microorganisms to produce lipid via metabolic engineering technology.

    PubMed

    Liang, Ming-Hua; Jiang, Jian-Guo

    2013-10-01

    With the depletion of global petroleum and its increasing price, biodiesel has been becoming one of the most promising biofuels for global fuels market. Researchers exploit oleaginous microorganisms for biodiesel production due to their short life cycle, less labor required, less affection by venue, and easier to scale up. Many oleaginous microorganisms can accumulate lipids, especially triacylglycerols (TAGs), which are the main materials for biodiesel production. This review is covering the related researches on different oleaginous microorganisms, such as yeast, mold, bacteria and microalgae, which might become the potential oil feedstocks for biodiesel production in the future, showing that biodiesel from oleaginous microorganisms has a great prospect in the development of biomass energy. Microbial oils biosynthesis process includes fatty acid synthesis approach and TAG synthesis approach. In addition, the strategies to increase lipids accumulation via metabolic engineering technology, involving the enhancement of fatty acid synthesis approach, the enhancement of TAG synthesis approach, the regulation of related TAG biosynthesis bypass approaches, the blocking of competing pathways and the multi-gene approach, are discussed in detail. It is suggested that DGAT and ME are the most promising targets for gene transformation, and reducing PEPC activity is observed to be beneficial for lipid production. Copyright © 2013 Elsevier Ltd. All rights reserved.

  16. Screening for microorganisms producing D-malate from maleate.

    PubMed Central

    van der Werf, M J; van den Tweel, W J; Hartmans, S

    1992-01-01

    More than 300 microorganisms were screened for their ability to convert maleate into D-malate as a result of the action of maleate hydratase. Accumulation of fumarate during incubation of permeabilized cells with maleate was shown to be indicative of one of the two enzymes known to transform maleate. The ratio in which fumarate and malate accumulated could be used to estimate the enantiomeric composition of the malate formed. Many strains (n = 128) were found to be capable of converting maleate to D-malate with an enantiomeric purity of more than 97%. Pseudomonas pseudoalcaligenes NCIMB 9867 was selected for more detailed studies. Although this strain was not able to grow on maleate, permeabilized cells were able to degrade maleate to undetectable levels, with a concomitant formation of D-malate. The D-malate was formed with an enantiomeric purity of more than 99.97%. PMID:1444397

  17. Organic acid-tolerant microorganisms and uses thereof for producing organic acids

    DOEpatents

    Pfleger, Brian Frederick; Begemann, Matthew Brett

    2014-05-06

    Organic acid-tolerant microorganisms and methods of using same. The organic acid-tolerant microorganisms comprise modifications that reduce or ablate AcsA activity or AcsA homolog activity. The modifications increase tolerance of the microorganisms to such organic acids as 3-hydroxypropionic acid (3HP), acrylic acid, and propionic acid. Further modifications to the microorganisms such as increasing expression of malonyl-CoA reductase and/or acetyl-CoA carboxylase provide or increase the ability of the microorganisms to produce 3HP. Methods of generating an organic acid with the modified microorganisms are provided. Methods of using acsA or homologs thereof as counter-selectable markers include replacing acsA or homologs thereof in cells with genes of interest and selecting for the cells comprising the genes of interest with amounts of organic acids effective to inhibit growth of cells harboring acsA or the homologs.

  18. Volatile dimethyl polonium produced by aerobic marine microorganisms.

    PubMed

    Bahrou, Andrew S; Ollivier, Patrick R L; Hanson, Thomas E; Tessier, Emmanuel; Amouroux, David; Church, Thomas M

    2012-10-16

    The production of volatile polonium (Po(v)), a naturally occurring radioactive element, by pure cultures of aerobic marine tellurite-resistant microorganisms was investigated. Rhodotorula mucilaginosa, a carotogenic yeast, and a Bacillus sp. strain, a Gram-positive bacterium, generated approximately one and 2 orders of magnitude, respectively, greater amounts of Po(v) compared to the other organisms tested. Gas chromatography-inductively coupled plasma-mass spectrometry (GC-ICP-MS) analysis identified dimethyl polonide (DMPo) as the predominant volatile Po compound in culture headspace of the yeast. This species assignment is based on the exact relation between GC retention times and boiling points of this and other Group VI B analogues (S, Se, and Te). The extent of the biotic Po(v) production correlates exponentially with elevated particulate Po (Po(p)): dissolved Po (Po(aq)) ratios in the cultures, consistent with efficient Po bioaccumulation. Further experimentation demonstrated that some abiotic Po(v) generation is possible. However, high-level Po(v) generation in these cultures is predominantly biotic.

  19. Volatiles produced by microorganisms isolated from refrigerated chicken at spoilage.

    PubMed Central

    Freeman, L R; Silverman, G J; Angelini, P; Merritt, C; Esselen, W B

    1976-01-01

    Volatile components present at spoilage of refrigerated chicken breasts were identified using high-vacuum-low-temperature distillation techniques followed by analysis with combined temperature-programmed gas chromatography and mass spectrometry. A comparison was made of the compounds detected from both irradiated and non-irradiated muscle stored at 2 and 10 degrees C under both aerobic and anaerobic conditions. Isolates were randomly selected from the spoiled poultry, identified, and evaluated for their ability to produce volatile spoilage noted when grown on radiation-sterilized chicken. Several isolates that produced off-odors on sterile chicken breasts were examined. Twenty-two compounds were associated with spoilage. Some of the compounds found on both irradiated and unirradiated samples were considered to play only a minor role in the spoilage aroma or were present in low concentrations, since the aroma of spoiled irradiated chicken lacked the harsh odor notes typical of spoiled unirradiated chicken. Fifteen of the 22 compounds were considered to be unique to unirradiated, aerobically spoiled samples. Nine of these compounds, hydrogen sulfide, methyl mercaptan, dimethyl sulfide, dimethyl disulfide, methyl acetate, ethyl acetate, heptadiene, methanol, and ethanol, were found on chicken spoiled at both 2 and 10 degrees C. xylene, benzaldehyde, and 2,3-dithiahexane were detected only in samples stored at 2 degrees C and methyl thiolacetate, 2-butanone, and ethyl propionate were associated with 10 degrees C spoilage. Fifty-eight isolates randomly selected from fresh, radiation-pasteurized, and unirradiated spoiled poultry were classified taxonomically, and 10 of them, which produced spoilage odors on sterilized chicken breasts, were selected for subsequent analysis of their volatiles. Isolates identified as Pseudomonas putrefaciens and Pseudomonas species that were members of groups I and II of Shewan's classification, as well as Flavobacterium and oxidative

  20. [PERSPECTIVES OF DEVELOPMENT OF LIVE RECOMBINANT ANTHRAX VACCINES BASED ON OPPORTUNISTIC AND APATHOGENIC MICROORGANISMS].

    PubMed

    Popova, P Yu; Mikshis, N I

    2016-01-01

    Live genetic engineering anthrax vaccines on the platform of avirulent and probiotic micro-organisms are a safe and adequate alternative to preparations based on attenuated Bacillus anthracis strains. Mucosal application results in a direct contact of the vaccine preparations with mucous membranes in those organs arid tissues of the macro-organisms, that are exposed to the pathogen in the first place, resulting in a development of local and systemic immune response. Live recombinant anthrax vaccines could be used both separately as well as in a prime-boost immunization scheme. The review focuses on immunogenic and protective properties of experimental live genetic engineering prearations, created based on members of geni of Salmonella, Lactobacillus and adenoviruses.

  1. Genetic structure of a novel biofuel-producing microorganism community.

    PubMed

    de Felice, Bruna; Blasi, Vito Onofrio; de Castro, Olga; Cennamo, Paola; Martino, Laura; Trifuoggi, Marco; Condorelli, Valerio; di Onofrio, Valeria; Guida, Marco

    2012-08-01

    Biofuels are an important alternative, renewable source of energy in the face of the ongoing depletion of fossil fuels. Cheese whey is a dairy industry waste characterized by high lactose concentration, which represents a significant environmental problem. Bio-ethanol production by cheese whey could be an effective nonvegetable source for renewable energy production. Here, we report the isolation of a mixed microbial population, able to produce ethanol as main fermentation product from fermenting whey. The microbial consortium has been used to perform a batch fermentation of crude whey in both anoxic and hypoxic conditions. Maximum ethanol concentrations achieved in this study was obtained using the mixed culture in hypoxic conditions, grown at pH 4 and 30 °C, with ethanol production yield of 60 g/L. Our research has pointed out an alternative way to both dispose and valorize cheese whey, a dairy by-product that could cause water pollution and harm to the environment if not properly treated.

  2. A search for microorganisms producing medium-chain alkanes from aldehydes.

    PubMed

    Ito, Masakazu; Kambe, Hiromi; Kishino, Shigenobu; Muramatsu, Masayoshi; Ogawa, Jun

    2017-08-28

    Microorganisms with medium-chain alkane-producing activity are promising for the bio-production of drop-in fuel. In this study, we screened for microorganisms producing tridecane from tetradecanal. The activity of aldehyde decarbonylation was found in a wide range of microbes. In particular, the genus Klebsiella in the Enterobacteriaceae family was found to have a high ability to produce alkanes from aldehydes via enzyme catalyzed reaction. Copyright © 2017 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.

  3. Biocidal Efficacy of Dissolved Ozone, Formaldehyde and Sodium Hypochlorite Against Total Planktonic Microorganisms in Produced Water

    NASA Astrophysics Data System (ADS)

    Puyate, Y. T.; Rim-Rukeh, A.

    The performance of three biocides (dissolved ozone, formaldehyde and sodium hypochlorite) in eliminating the bacteria and fungi in produced water is investigated experimentally. The analysis involves monitoring the microbial population in nine conical flasks each containing the same volume of a mixture of produced water, culture medium that sustains the growth of microorganisms and a known concentration of biocide. The concentrations of each biocide used in the study are 0.1, 0.2 and 0.5 ppm. It is shown that dissolved ozone exhibits the best biocidal characteristics and a concentration of 0.5 ppm eliminated all the microorganisms in the produced water after 150 min contact time.

  4. Insecticidal activity of recombinant avidin produced in yeast.

    PubMed

    Hinchliffe, Gareth; Bown, David P; Gatehouse, John A; Fitches, Elaine

    2010-06-01

    An expression construct encoding chicken (Gallus gallus) avidin was assembled from amplified fragments of genomic DNA. Recombinant, functional avidin was produced in Pichia pastoris, with yields of up to 80 mg/l of culture supernatant. The recombinant avidin had similar insecticidal activity to egg white avidin when assayed against larvae of a lepidopteran crop pest, cabbage moth (Mamestra brassicae), causing >90% reduction in growth and 100% mortality when fed in optimised diets at levels of 1.5 microM and 15 microM (100 ppm and 1000 ppm wet weight of recombinant protein). The recombinant protein was also highly toxic to a hemipteran pest, the pea aphid (Acyrthosiphon pisum), when fed in liquid artificial diet, causing 100% mortality after 4 days when present at concentrations > or = 3.8 microM (0.25 mg/ml, 250 ppm). Mortality was dose-dependent, with an estimated LC(50) of 2.1 microM. Toxicity to A. pisum was prevented by biotin supplementation of diet. In contrast, avidin had no significant effects on the survival of cereal aphid (Sitobion avenae) at concentrations up to 30 microM in liquid diet. Analysis of genomic DNA showed that symbionts from both aphid species lack the ability to synthesise biotin de novo. Cereal aphids appear to be less sensitive to recombinant avidin in the diet through proteolysis of the ingested protein, which would allow recovery of bound biotin. Crown Copyright 2010. Published by Elsevier Ltd. All rights reserved.

  5. Functional protein-based nanomaterial produced in microorganisms recognized as safe: A new platform for biotechnology.

    PubMed

    Cano-Garrido, Olivia; Sánchez-Chardi, Alejandro; Parés, Sílvia; Giró, Irene; Tatkiewicz, Witold I; Ferrer-Miralles, Neus; Ratera, Imma; Natalello, Antonino; Cubarsi, Rafael; Veciana, Jaume; Bach, Àlex; Villaverde, Antonio; Arís, Anna; Garcia-Fruitós, Elena

    2016-10-01

    Inclusion bodies (IBs) are protein-based nanoparticles formed in Escherichia coli through stereospecific aggregation processes during the overexpression of recombinant proteins. In the last years, it has been shown that IBs can be used as nanostructured biomaterials to stimulate mammalian cell attachment, proliferation, and differentiation. In addition, these nanoparticles have also been explored as natural delivery systems for protein replacement therapies. Although the production of these protein-based nanomaterials in E. coli is economically viable, important safety concerns related to the presence of endotoxins in the products derived from this microorganism need to be addressed. Lactic acid bacteria (LAB) are a group of food-grade microorganisms that have been classified as safe by biologically regulatory agencies. In this context, we have demonstrated herein, for the first time, the production of fully functional, IB-like protein nanoparticles in LAB. These nanoparticles have been fully characterized using a wide range of techniques, including field emission scanning electron microscopy (FESEM), transmission electron microscopy (TEM), dynamic light scattering (DLS), Fourier transform infrared (FTIR) spectroscopy, zymography, cytometry, confocal microscopy, and wettability and cell coverage measurements. Our results allow us to conclude that these materials share the main physico-chemical characteristics with IBs from E. coli and moreover are devoid of any harmful endotoxin contaminant. These findings reveal a new platform for the production of protein-based safe products with high pharmaceutical interest. The development of both natural and synthetic biomaterials for biomedical applications is a field in constant development. In this context, E. coli is a bacteria that has been widely studied for its ability to naturally produce functional biomaterials with broad biomedical uses. Despite being effective, products derived from this species contain membrane

  6. Metabolic engineering of microorganisms to produce omega-3 very long-chain polyunsaturated fatty acids.

    PubMed

    Gong, Yangmin; Wan, Xia; Jiang, Mulan; Hu, Chuanjiong; Hu, Hanhua; Huang, Fenghong

    2014-10-01

    Omega-3 long-chain polyunsaturated fatty acids (LC-PUFAs) have received growing attention due to their significant roles in human health. Currently the main source of these nutritionally and medically important fatty acids is marine fish, which has not met ever-increasing global demand. Microorganisms are an important alternative source also being explored. Although many microorganisms accumulate omega-3 LC-PUFAs naturally, metabolic engineering might still be necessary for significantly improving their yields. Here, we review recent research involving the engineering of microorganisms for production of omega-3 LC-PUFAs, including eicospentaenoic acid and docosohexaenoic acid. Both reconstitution of omega-3 LC-PUFA biosynthetic pathways and modification of existing pathways in microorganisms have demonstrated the potential to produce high levels of omega-3 LC-PUFAs. However, the yields of omega-3 LC-PUFAs in host systems have been substantially limited by potential metabolic bottlenecks, which might be caused partly by inefficient flux of fatty acid intermediates between the acyl-CoA and different lipid class pools. Although fatty acid flux in both native and heterologous microbial hosts might be controlled by several acyltransferases, evidence has suggested that genetic manipulation of one acyltransferase alone could significantly increase the accumulation of LC-PUFAs. The number of oleaginous microorganisms that can be genetically transformed is increasing, which will advance engineering efforts to maximize LC-PUFA yields in microbial strains.

  7. Effect of enzyme producing microorganisms on the biomass of epigeic earthworms (eisenia fetida) in vermicompost.

    PubMed

    Hong, Sung Wook; Lee, Ju Sam; Chung, Kun Sub

    2011-05-01

    We analyzed the bacterial community structure of the intestines of earthworms and determined the effect of enzyme producing microorganisms on the biomass of earthworms in vermicompost. Fifty-seven bacterial 16S rDNA clones were identified in the intestines of earthworms by using polymerase chain reaction-denaturing gradient gel electrophoresis (PCR-DGGE) analysis. Entomoplasma somnilux and Bacillus licheniformis were the dominant microorganisms; other strains included Aeromonas, Bacillus, Clostridium, Ferrimonas, and uncultured bacteria. Among these strains, Photobacterium ganghwense, Aeromonas hydrophila, and Paenibacillus motobuensis were enzyme-producing microorganisms. In the mixtures that were inoculated with pure cultures of A. hydrophila WA40 and P. motobuensis WN9, the highest survival rate was 100% and the average number of earthworms, young earthworms, and cocoons were 10, 4.00-4.33, and 3.00-3.33, respectively. In addition, P. motobuensis WN9 increased the growth of earthworms and production of casts in the vermicompost. These results show that earthworms and microorganisms have a symbiotic relationship.

  8. [Research on search of the carotenoid-producing microorganisms in marine area and the improvement of production ratio].

    PubMed

    Sakagami, Yoshikazu; Sumiya, Yasuji; Komemushi, Sadawo

    2010-11-01

    Carotenoids are liposoluble pigments widely distributed in nature. More than 750 carotenoids are isolated from natural sources, but only a few kinds are used industrially. The production of carotenoid by microorganisms is to be expected, but few carotenoids originate from living things on land. And there is little knowledge about carotenoid-producing microorganisms in the oceans. The possibility still exists of discovering new carotenoid-producing microorganisms. Sunlight is very strong in subtropical regions. The surface of the sea and coral reefs in these regions is a severe environment for growth of microorganisms. While such conditions produce reactive oxygen species, the continuing strong irradiation can also lead to damaging and lethal photo-oxidative reactions. Many undiscovered microorganisms may possess protective mechanisms such as anti-oxidative activities for survival in this environment. This study focused on marine microorganisms inhabiting coral reefs in the Okinawa area, especially carotenoid-producing bacteria possessing anti-oxidative activities. Many carotenoid-producing microorganisms were collected from subtropical ocean areas (a total of 334 strains of pigmented microorganisms), and the chemical composition, some culture conditions and genetic characteristics of the carotenoids from these microorganisms were examined. Furthermore, similar research was performed using some creatures from the ocean surrounding Kochi Prefecture.

  9. Achievements and perspectives to overcome the poor solvent resistance in acetone and butanol-producing microorganisms.

    PubMed

    Ezeji, Thaddeus; Milne, Caroline; Price, Nathan D; Blaschek, Hans P

    2010-02-01

    Anaerobic bacteria such as the solventogenic clostridia can ferment a wide range of carbon sources (e.g., glucose, galactose, cellobiose, mannose, xylose, and arabinose) to produce carboxylic acids (acetic and butyric) and solvents such as acetone, butanol, and ethanol (ABE). The fermentation process typically proceeds in two phases (acidogenic and solventogenic) in a batch mode. Poor solvent resistance by the solventogenic clostridia and other fermenting microorganisms is a major limiting factor in the profitability of ABE production by fermentation. The toxic effect of solvents, especially butanol, limits the concentration of these solvents in the fermentation broth, limiting solvent yields and adding to the cost of solvent recovery from dilute solutions. The accepted dogma is that toxicity in the ABE fermentation is due to chaotropic effects of butanol on the cell membranes of the fermenting microorganisms, which poses a challenge for the biotechnological whole-cell bio-production of butanol. This mini-review is focused on (1) the effects of solvents on inhibition of cell metabolism (nutrient transport, ion transport, and energy metabolism); (2) cell membrane fluidity, death, and solvent tolerance associated with the ability of cells to tolerate high concentrations of solvents without significant loss of cell function; and (3) strategies for overcoming poor solvent resistance in acetone and butanol-producing microorganisms.

  10. Cellulase-containing cell-free fermentate produced from microorganism ATCC 55702

    DOEpatents

    Dees, H. Craig

    1997-12-16

    Bacteria which produce large amounts of cellulase-containing cell-free fermentate have been identified. The original bacterium (ATCC 55703) was genetically altered using nitrosoguanidine (MNNG) treatment to produce the enhanced cellulase producing bacterium (ATCC 55702), which was identified through replicate plating. ATCC 55702 has improved characteristics and qualities for the degradation of cellulosic waste materials for fuel production, food processing, textile processing, and other industrial applications. ATCC 55702 is an improved bacterial host for genetic manipulations using recombinant DNA techniques, and is less likely to destroy genetic manipulations using standard mutagenesis techniques.

  11. Cellulase-containing cell-free fermentate produced from microorganism ATCC 55702

    DOEpatents

    Dees, H.C.

    1997-12-16

    Bacteria which produce large amounts of cellulase-containing cell-free fermentate have been identified. The original bacterium (ATCC 55703) was genetically altered using nitrosoguanidine (MNNG) treatment to produce the enhanced cellulase producing bacterium (ATCC 55702), which was identified through replicate plating. ATCC 55702 has improved characteristics and qualities for the degradation of cellulosic waste materials for fuel production, food processing, textile processing, and other industrial applications. ATCC 55702 is an improved bacterial host for genetic manipulations using recombinant DNA techniques, and is less likely to destroy genetic manipulations using standard mutagenesis techniques. 5 figs.

  12. Aquatic Plant Control Research Program. Biological Control of Hydrilla verticillata (L.f.) Royle with Lytic Enzyme-Producing Microorganisms.

    DTIC Science & Technology

    1985-09-01

    Lytic enzyme-producing microorganisms Biocontrol Mycoherbicides Hydrilla Induced pathogenicity 20. ASTRACT (Coartinue G rev’wm eft if n*..eeam7 mod...However, no natural enemies of hydrilla have yet been imported that are promising biocontrol candidates. Therefore, a less conventional approach was...of microorganisms that function in the decay process. These microorganisms pro- duce enzymes capable of lysing specific plant components such as

  13. [Characteristics of the response of natural and recombinant luminescent microorganisms in the presence of Fe2+ ions].

    PubMed

    Deriabin, D G; Karimov, I F

    2010-01-01

    It was found that divalent iron ions have alternative effects on the bioluminescence of the natural marine microorganism Photobacterium phosphoreum and the recombinant Escherichia coli strain with a cloned lux operon of P. leiognathi. In the presence of 0.25-5.0 mM FeSO4, the bioluminescence intensity of the former and the latter increased and decreased, respectively. To establish the causes of these differences, we studied the characteristics of the fatty acid composition of the compared microorganisms. The fatty acid profile of E. coli was characterized by a high proportion of unsaturated 11-octadecenoic (vaccenic) acid. A study of this acid in a cell-free enzyme system used for bioluminescence generation showed that it is a potent inhibitor of bacterial bioluminescence. It was found that such effects are enhanced if 11-octadecenoid acid is preincubated with Fe2+.

  14. [A new plate method for screening of polysaccharide-degrading enzymes and their producing microorganisms].

    PubMed

    Ma, Xiang-Dong; Ke, Tao; Xiong, Lan; Yan, Hong; Ma, Li-Xin

    2007-12-01

    A plate assay based on the formation of haloes on Petri dishes, containing the trypan blue dye and polysaccharides as substrates, provides a specific, reliable and rapid detection of corresponding polysaccharide degrading enzymes and their producing microorganisms. A blue complex was formed by mixing trypan blue and polysaccharides as substrates. It has been proved by testing three strains that the trypan blue was neither harmful to microorganisms nor enzymes and could stand the normal sterilization. It's optimum concentration was from 0.005% to 0.01% (W/V). It do not need to prepare dye-labelled polysaccharides, so is a money and time-consuming method. The sensitivity of trypan blue method was the same as traditional method and it has potential for increasing the efficacy of screening of microorganisms, utilizing different polysaccharides, especially for large-scale searching programs, such as screening of large numbers of natural samples and engineering bacteria. Using this method, polysaccharide-degrading enzyme genes also has potential of as a new kind of marker gene in gene engineering techniques. According to the result, this method is suitable for detecting cellulase, amylase, pullulanase and mannase, but not suitable for detecting xylanase and inulinase.

  15. Quality assessment of recombinant proteins produced in plants.

    PubMed

    Medrano, Giuliana; Dolan, Maureen C; Condori, Jose; Radin, David N; Cramer, Carole L

    2012-01-01

    Plant-based expression technologies for recombinant proteins have begun to receive acceptance for pharmaceuticals and other commercial markets. Protein products derived from plants offer safer, more cost-effective, and less capital-intensive alternatives to traditional manufacturing systems using microbial fermentation or animal cell culture bioreactors. Moreover, plants are now known to be capable of expressing bioactive proteins from a diverse array of species including animals and humans. Methods development to assess the quality and performance of proteins manufactured in plants are essential to support the QA/QC demands as plant-produced protein products transition to the commercial marketplace. Within the pharmaceutical arena, process validation and acceptance criteria for biological products must comply with the Food and Drug Administration (FDA) and ICH Q6B guidelines in order to initiate the regulatory approval process. Detailed product specifications will also need to be developed and validated for plant-made proteins for the bioenergy, food, chemical synthesis, or research reagent markets.We have, therefore, developed assessment methods for important qualitative and quantitative parameters of the products and the manufacturing methods utilized in plant-based production systems. In this chapter, we describe a number of procedures to validate product identity and characteristics including mass analyses, antibody cross-reactivity, N-terminal sequencing, and bioactivity. We also address methods for routine assessment of yield, recovery, and purity. The methods presented are those developed for the synthesis and recovery of the avian cytokine, chicken interleukin-12 (ChIL-12), produced in the leaves of Nicotiana benthamiana. The ChIL-12 protein used as a model for this chapter includes a C-terminal histidine epitope (HIS-tag) and, thus, these methods may be directly applicable to other HIS-tagged proteins produced in plants. However, the overall strategy

  16. Lactogenic immunity in transgenic mice producing recombinant antibodies neutralizing coronavirus.

    PubMed

    Castilla, J; Sola, I; Pintado, B; Sánchez-Morgado, J M; Enjuanes, L

    1998-01-01

    Protection against coronavirus infections can be provided by the oral administration of virus neutralizing antibodies. To provide lactogenic immunity, eighteen lines of transgenic mice secreting a recombinant IgG1 monoclonal antibody (rIgG1) and ten lines of transgenic mice secreting recombinant IgA monoclonal antibodies (rIgA) neutralizing transmissible gastroenteritis coronavirus (TGEV) into the milk were generated. Genes encoding the light and heavy chains of monoclonal antibody (MAb) 6A.C3 were expressed under the control of regulatory sequences derived from the mouse genomic DNA encoding the whey acidic protein (WAP) and beta-lactoglobulin (BLG), which are highly abundant milk proteins. The MAb 6A.C3 binds to a highly conserved epitope present in coronaviruses of several species. This MAb does not allow the selection of neutralization escaping virus mutants. The antibody was expressed in the milk of transgenic mice with titers of one million as determined by RIA, and neutralized TGEV infectivity by one million fold corresponding to immunoglobulin concentrations of 5 to 6 mg per ml. Matrix attachment regions (MAR) sequences were not essential for rIgG1 transgene expression, but co-microinjection of MAR and antibody genes led to a twenty to ten thousand-fold increase in the antibody titer in 50% of the rIgG1 transgenic animals generated. Co-microinjection of the genomic BLG gene with rIgA light and heavy chain genes led to the generation of transgenic mice carrying the three transgenes. The highest antibody titers were produced by transgenic mice that had integrated the antibody and BLG genes, although the number of transgenic animals generated does not allow a definitive conclusion on the enhancing effect of BLG co-integration. Antibody expression levels were transgene copy number independent and integration site dependent. The generation of transgenic animals producing virus neutralizing antibodies in the milk could be a general approach to provide protection

  17. [Biosynthesis and isolation of a recombinant protein for producing genetically-engineered human proinsulin].

    PubMed

    Ivankin, A N; Mitaleva, S I; Nekliudov, A D

    1998-01-01

    Isolation of the recombinant protein from a genetically engineered Escherichia coli 1854 producer for further chemical enzymatic transformation into human insulin through proinsulin was studied. Under optimal conditions, the recombinant protein formation was more than 35% of the total cell proteins. Structures of the polypeptides obtained and purified chromatographically were confirmed by amino acid analysis. Human proinsulin was derived from the recombinant protein isolated.

  18. Improved method for effective screening of ACC (1-aminocyclopropane-1-carboxylate) deaminase producing microorganisms.

    PubMed

    Patil, Chandrashekhar; Suryawanshi, Rahul; Koli, Sunil; Patil, Satish

    2016-12-01

    Aminocyclopropane-1-carboxylate deaminase (ACCD) producing microorganisms support plant growth under a variety of biotic and abiotic stress conditions such as drought, soil salinity, flooding, heavy metal pollution and phyto-pathogen attack. Available screening methods for ACCD give idea only about its primary microbial ACCD activity than the actual potential. In the present investigation, we have simply improved screening method by incorporating pH indicator dyes (phenol red and bromothymol blue) in ACC containing medium. This modification is based on the basic principle that ACCD action releases ammonia which can be detected by color change and zone around the bacterial colony. High color intensity and zone around the colony indicates most potent producer, colony showing only a color change indicates moderate potential and no change in colony color indicates least efficiency. Enzymatic bioassays as well as root elongation studies revealed that ACC-deaminase activity of Pseudomonas aeruginosa, Stenotrophomonas maltophilia and Bacillus subtilis clearly corresponds to their growth on dye incorporated ACC medium. This method could be used to complement the existing screening methods and to speed up the targeted isolation of agriculturally important microorganisms.

  19. High value added lipids produced by microorganisms: a potential use of sugarcane vinasse.

    PubMed

    Fernandes, Bruna Soares; Vieira, João Paulo Fernandes; Contesini, Fabiano Jares; Mantelatto, Paulo Eduardo; Zaiat, Marcelo; Pradella, José Geraldo da Cruz

    2017-12-01

    This review aims to present an innovative concept of high value added lipids produced by heterotrophic microorganisms, bacteria and fungi, using carbon sources, such as sugars, acids and alcohols that could come from sugarcane vinasse, which is the main byproduct from ethanol production that is released in the distillation step. Vinasse is a rich carbon source and low-cost feedstock produced in large amounts from ethanol production. In 2019, the Brazilian Ministry of Agriculture, Livestock and Food Supply estimates that growth of ethanol domestic consumption will be 58.8 billion liters, more than double the amount in 2008. This represents the annual production of more than 588 billion liters of vinasse, which is currently used as a fertilizer in the sugarcane crop, due to its high concentration of minerals, mainly potassium. However, studies indicate some disadvantages such as the generation of Greenhouse Gas emission during vinasse distribution in the crop, as well as the possibility of contaminating the groundwater and soil. Therefore, the development of programs for sustainable use of vinasse is a priority. One profitable alternative is the fermentation of vinasse, followed by an anaerobic digester, in order to obtain biomaterials such as lipids, other byproducts, and methane. Promising high value added lipids, for instance carotenoids and polyunsaturated fatty acids (PUFAS), with a predicted market of millions of US$, could be produced using vinasse as carbon source, to guide an innovative concept for sustainable production. Example of lipids obtained from the fermentation of compounds present in vinasse are vitamin D, which comes from yeast sucrose fermentation and Omega 3, which can be obtained by bacteria and fungi fermentation. Additionally, several other compounds present in vinasse can be used for this purpose, including sucrose, ethanol, lactate, pyruvate, acetate and other carbon sources. Finally, this paper illustrates the potential market and

  20. Uptake and degradation of discharged produced water components in marine microorganisms

    SciTech Connect

    Brakstad, O.G.; Olsen, A.J.; Nordtug, T.

    1996-12-31

    Produced waters from offshore oil production are a significant source of aromatic compounds discharged to the seawater. Exposure studies have revealed toxic effects of alkylated phenols and PAH compounds to various marine organisms. In this study the fate of aromatic compounds in seawater was investigated, using a dynamic exposure system which simulated dilution effects of discharged chemicals and {open_quotes}natural{close_quotes} conditions in the seawater recipient. {sup 14}C-labelled alkylated phenols (para-cresol) and polyaromatic hydrocarbons (PAH; naphthalene or phenanthrene) were applied to exposure tanks at sub-ppb concentrations by the aid of a computer-controlled injector device. Natural seawater, with normal seawater bacteria, cultures of the phytoplankton Isochrysis galbana, or the ciliate Euplotes bisulcatus, passed the exposure system at a residence time of approximately 5 hours, creating a short and defined exposure time between compounds and microorganisms. Compounds bound to or taken up by the organisms were collected on filters downstream the exposure system. The results showed that marine microorganisms may take up portions of aromatic compounds within a short period of time. Uptake mechanisms were expected to be passive events. Comparison of bioconcentration factors to the water-octanol coefficients of the components indicated alternative uptake mechanisms to a passive incorporation in the lipid membranes of the organisms. Binding to surface protein and carbohydrate moieties may play a central role during uptake. Studies in static systems with exposure of components to normal seawater bacteria showed a significant uptake and mineralization only for p-cresol. Standard seawater BOD testing indicated that all compounds tested were potentially biodegradable in normal non-acclimated seawater. The results demonstrate that uptake and degradation of produced water components are important to consider during studies of the fate of these components.

  1. Reduction of butyrate- and methane-producing microorganisms in patients with Irritable Bowel Syndrome.

    PubMed

    Pozuelo, Marta; Panda, Suchita; Santiago, Alba; Mendez, Sara; Accarino, Anna; Santos, Javier; Guarner, Francisco; Azpiroz, Fernando; Manichanh, Chaysavanh

    2015-08-04

    The pathophysiology of irritable bowel syndrome (IBS) remains unclear. Here we investigated the microbiome of a large cohort of patients to identify specific signatures for IBS subtypes. We examined the microbiome of 113 patients with IBS and 66 healthy controls. A subset of these participants provided two samples one month apart. We analyzed a total of 273 fecal samples, generating more than 20 million 16S rRNA sequences. In patients with IBS, a significantly lower microbial diversity was associated with a lower relative abundance of butyrate-producing bacteria (P = 0.002; q < 0.06), in particular in patients with IBS-D and IBS-M. IBS patients who did not receive any treatment harboured a lower abundance of Methanobacteria compared to healthy controls (P = 0.005; q = 0.05). Furthermore, significant correlations were observed between several bacterial taxa and sensation of flatulence and abdominal pain (P < 0.05). Altogether, our findings showed that IBS-M and IBS-D patients are characterized by a reduction of butyrate producing bacteria, known to improve intestinal barrier function, and a reduction of methane producing microorganisms a major mechanism of hydrogen disposal in the human colon, which could explain excess of abdominal gas in IBS.

  2. Reduction of butyrate- and methane-producing microorganisms in patients with Irritable Bowel Syndrome

    PubMed Central

    Pozuelo, Marta; Panda, Suchita; Santiago, Alba; Mendez, Sara; Accarino, Anna; Santos, Javier; Guarner, Francisco; Azpiroz, Fernando; Manichanh, Chaysavanh

    2015-01-01

    The pathophysiology of irritable bowel syndrome (IBS) remains unclear. Here we investigated the microbiome of a large cohort of patients to identify specific signatures for IBS subtypes. We examined the microbiome of 113 patients with IBS and 66 healthy controls. A subset of these participants provided two samples one month apart. We analyzed a total of 273 fecal samples, generating more than 20 million 16S rRNA sequences. In patients with IBS, a significantly lower microbial diversity was associated with a lower relative abundance of butyrate-producing bacteria (P = 0.002; q < 0.06), in particular in patients with IBS-D and IBS-M. IBS patients who did not receive any treatment harboured a lower abundance of Methanobacteria compared to healthy controls (P = 0.005; q = 0.05). Furthermore, significant correlations were observed between several bacterial taxa and sensation of flatulence and abdominal pain (P < 0.05). Altogether, our findings showed that IBS-M and IBS-D patients are characterized by a reduction of butyrate producing bacteria, known to improve intestinal barrier function, and a reduction of methane producing microorganisms a major mechanism of hydrogen disposal in the human colon, which could explain excess of abdominal gas in IBS. PMID:26239401

  3. Evaluation of terrestrial microcosms for detection, fate, and survival analysis of genetically engineered microorganisms and their recombinant genetic material

    SciTech Connect

    Fredrickson, J.K.; Seidler, R.J.

    1989-02-01

    The research included in this document represents the current scientific information available regarding the applicability of terrestrial microcosms and related methodologies for evaluating detection methods and the fate and survival of microorganisms in the environment. The three terrestrial microcosms described in this document were used to evaluate the survival and fate of recombinant bacteria in soils and in association with plant surfaces and insects and their transport through soil with percolating water and root systems, and to test new methods and procedures to improve detection and enumeration of bacteria in soil. Simple (potting soil composed of peat mix and perlite, lacking environmental control and monitoring) and complex microcosms (agricultural soil with partial control and monitoring of environmental conditions) were demonstrated to be useful tools for preliminary assessments of microbial viability in terrestrial ecosystems. These studies evaluated the survival patterns of Enterobacter cloacae (pBR322) in soil and on plant surfaces and the ingestion of this same microorganism by cutworms and survival in the foregut and frass. The Versacore microcosm design was used to monitor the fate and competitiveness of genetically engineered bacteria in soil. Both selective media and gene probes were used successfully to follow the fate of two recombinant Pseudomonas sp. introduced into Versacore microcosms. Intact soil-core microcosms were employed to evaluate the fate and transport of genetically altered Azospirillum sp. and Pseudomonas sp. in soil and the plant rhizosphere. The usefulness of these various microcosms as a tool for risk assessment is underscored by the ease in obtaining soil from a proposed field release site to evaluate subsequent GEM fate and survival.

  4. Vaccinia virus vectors: new strategies for producing recombinant vaccines.

    PubMed Central

    Hruby, D E

    1990-01-01

    The development and continued refinement of techniques for the efficient insertion and expression of heterologous DNA sequences from within the genomic context of infectious vaccinia virus recombinants are among the most promising current approaches towards effective immunoprophylaxis against a variety of protozoan, viral, and bacterial human pathogens. Because of its medical relevance, this area is the subject of intense research interest and has evolved rapidly during the past several years. This review (i) provides an updated overview of the technology that exists for assembling recombinant vaccinia virus strains, (ii) discusses the advantages and disadvantages of these approaches, (iii) outlines the areas of outgoing research directed towards overcoming the limitations of current techniques, and (iv) provides some insight (i.e., speculation) about probable future refinements in the use of vaccinia virus as a vector. PMID:2187593

  5. Method for producing aldehyde from CO.sub.2

    SciTech Connect

    Liao, James C.; Atsumi, Shota

    2015-09-29

    The invention provides recombinant microorganisms capable of producing isobutyraldehyde using CO.sub.2 as a carbon source. The invention further provides methods of preparing and using such microorganisms to produce isobutyraldehyde.

  6. Human recombinant type I collagen produced in plants.

    PubMed

    Shoseyov, Oded; Posen, Yehudit; Grynspan, Frida

    2013-07-01

    As a central element of the extracellular matrix, collagen is intimately involved in tissue development, remodeling, and repair and confers high tensile strength to tissues. Numerous medical applications, particularly, wound healing, cell therapy, bone reconstruction, and cosmetic technologies, rely on its supportive and healing qualities. Its synthesis and assembly require a multitude of genes and post-translational modifications, where even minor deviations can be deleterious or even fatal. Historically, collagen was always extracted from animal and human cadaver sources, but bare risk of contamination and allergenicity and was subjected to harsh purification conditions resulting in irreversible modifications impeding its biofunctionality. In parallel, the highly complex and stringent post-translational processing of collagen, prerequisite of its viability and proper functioning, sets significant limitations on recombinant expression systems. A tobacco plant expression platform has been recruited to effectively express human collagen, along with three modifying enzymes, critical to collagen maturation. The plant extracted recombinant human collagen type I forms thermally stable helical structures, fibrillates, and demonstrates bioactivity resembling that of native collagen. Deployment of the highly versatile plant-based biofactory can be leveraged toward mass, rapid, and low-cost production of a wide variety of recombinant proteins. As in the case of collagen, proper planning can bypass plant-related limitations, to yield products structurally and functionally identical to their native counterparts.

  7. [Dependence of peripheral blood lymphocyte subpopulations on causative microorganisms able to produce superantigens].

    PubMed

    Verba, Vytis; Gudzinskiene, Solveiga

    2002-01-01

    A retrospective study of 176 immunologically tested patients admitted to Kaunas Medical University Hospital during 1997-2000 was performed. All patients had positive bacteriological culture test result confirming an infectious etiology of the disease. Our results showed that majority of immunological parameters were dependent on such non-specific factors as intensity and localization of the inflammatory process, an overall functional status of the patient, and the number of the disease exacerbation episodes during the last year before admission. In contrast to this, the absolute number of CD4 lymphocytes, the relative amount of HLA-DR positive lymphocytes and the index of neutrophil latex phagocytosis were exceptionally dependent on the species of the causative microorganism, in particular on superantigen producing cocci. In this case, the HLA-DR positive lymphocyte amount and the neutrophil phagocytosis index were significantly higher. In addition, the CD4/CD8 lymphocyte ratio (the immunoregulatory index) was significantly lower in this group. As much as those findings are concordant with the signs of excessive immune activation, we conclude that they reflect a possible superantigenic action of the disease causing bacteria. Therefore, a need for immunomodulating therapy during the infections caused by species able to produce superantigens is confirmed.

  8. Conversion of sucrose into isomaltulose by Enterobacter sp. FMB1, an isomaltulose-producing microorganism isolated from traditional Korean food.

    PubMed

    Cho, Mee-Hyun; Park, Sang-Eun; Lim, Jin Kyu; Kim, Jong-Sang; Kim, Jeong Hwan; Kwon, Dae Young; Park, Cheon-Seok

    2007-03-01

    Over 500 microorganisms isolated from Korean traditional foods, Maeju (source of soybean paste) and Nuruk (Korean koji), were screened to obtain an isomaltulose-producing microorganism. It was identified as Enterobacter sp. FMB-1 by 16S rRNA sequencing and the API 20E system. It had a greater than 90% conversion of sucrose (as 4 g/l) to isomaltulose in 2 days. Small amounts of trehalulose, glucose, and fructose were produced as byproducts, implying that this strain could be possibly employed in the production of isomaltulose in industry.

  9. Controlled glycosylation of plant-produced recombinant proteins.

    PubMed

    Strasser, Richard; Altmann, Friedrich; Steinkellner, Herta

    2014-12-01

    Despite their recognized importance for therapeutic proteins, the production of structurally defined glycans is still a challenging issue. However, an increased understanding of glycosylation pathways, recent advances in analytical tools, and emerging technologies for subcellular targeting using chimeric glycosyltransferases are facilitating the rational design of new glycan biosynthetic pathways. Plants are particularly amenable to glyco-engineering approaches and thus they are increasingly being used for the production of recombinant proteins. Here we summarize the main achievements in the field of in planta glyco-engineering for the production of therapeutically relevant proteins. Copyright © 2014 Elsevier Ltd. All rights reserved.

  10. 21 CFR 878.4494 - Absorbable poly(hydroxybutyrate) surgical suture produced by recombinant DNA technology.

    Code of Federal Regulations, 2013 CFR

    2013-04-01

    ... produced by recombinant DNA technology. 878.4494 Section 878.4494 Food and Drugs FOOD AND DRUG... recombinant DNA technology. (a) Identification. An absorbable poly(hydroxybutyrate) surgical suture is an... deoxyribonucleic acid (DNA) technology. The device is intended for use in general soft tissue approximation and...

  11. 21 CFR 878.4494 - Absorbable poly(hydroxybutyrate) surgical suture produced by recombinant DNA technology.

    Code of Federal Regulations, 2014 CFR

    2014-04-01

    ... produced by recombinant DNA technology. 878.4494 Section 878.4494 Food and Drugs FOOD AND DRUG... recombinant DNA technology. (a) Identification. An absorbable poly(hydroxybutyrate) surgical suture is an... deoxyribonucleic acid (DNA) technology. The device is intended for use in general soft tissue approximation and...

  12. 21 CFR 878.4494 - Absorbable poly(hydroxybutyrate) surgical suture produced by recombinant DNA technology.

    Code of Federal Regulations, 2012 CFR

    2012-04-01

    ... produced by recombinant DNA technology. 878.4494 Section 878.4494 Food and Drugs FOOD AND DRUG... recombinant DNA technology. (a) Identification. An absorbable poly(hydroxybutyrate) surgical suture is an... deoxyribonucleic acid (DNA) technology. The device is intended for use in general soft tissue approximation and...

  13. 21 CFR 878.4494 - Absorbable poly(hydroxybutyrate) surgical suture produced by recombinant DNA technology.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... produced by recombinant DNA technology. 878.4494 Section 878.4494 Food and Drugs FOOD AND DRUG... recombinant DNA technology. (a) Identification. An absorbable poly(hydroxybutyrate) surgical suture is an... deoxyribonucleic acid (DNA) technology. The device is intended for use in general soft tissue approximation and...

  14. 21 CFR 878.4494 - Absorbable poly(hydroxybutyrate) surgical suture produced by recombinant DNA technology.

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    ... produced by recombinant DNA technology. 878.4494 Section 878.4494 Food and Drugs FOOD AND DRUG... recombinant DNA technology. (a) Identification. An absorbable poly(hydroxybutyrate) surgical suture is an... deoxyribonucleic acid (DNA) technology. The device is intended for use in general soft tissue approximation and...

  15. The role of microaerophilic Fe-oxidizing micro-organisms in producing banded iron formations.

    PubMed

    Chan, C S; Emerson, D; Luther, G W

    2016-09-01

    Despite the historical and economic significance of banded iron formations (BIFs), we have yet to resolve the formation mechanisms. On modern Earth, neutrophilic microaerophilic Fe-oxidizing micro-organisms (FeOM) produce copious amounts of Fe oxyhydroxides, leading us to wonder whether similar organisms played a role in producing BIFs. To evaluate this, we review the current knowledge of modern microaerophilic FeOM in the context of BIF paleoenvironmental studies. In modern environments wherever Fe(II) and O2 co-exist, microaerophilic FeOM proliferate. These organisms grow in a variety of environments, including the marine water column redoxcline, which is where BIF precursor minerals likely formed. FeOM can grow across a range of O2 concentrations, measured as low as 2 μm to date, although lower concentrations have not been tested. While some extant FeOM can tolerate high O2 concentrations, many FeOM appear to prefer and thrive at low O2 concentrations (~3-25 μm). These are similar to the estimated dissolved O2 concentrations in the few hundred million years prior to the 'Great Oxidation Event' (GOE). We compare biotic and abiotic Fe oxidation kinetics in the presence of varying levels of O2 and show that microaerophilic FeOM contribute substantially to Fe oxidation, at rates fast enough to account for BIF deposition. Based on this synthesis, we propose that microaerophilic FeOM were capable of playing a significant role in depositing the largest, most well-known BIFs associated with the GOE, as well as afterward when global O2 levels increased.

  16. A Highly Efficient and Simple Construction Strategy for Producing Recombinant Baculovirus Bombyx mori Nucleopolyhedrovirus

    PubMed Central

    Liu, Xingjian; Wei, Yonglong; Li, Yinü; Li, Haoyang; Yang, Xin; Yi, Yongzhu; Zhang, Zhifang

    2016-01-01

    The silkworm baculovirus expression system is widely used to produce recombinant proteins. Several strategies for constructing recombinant viruses that contain foreign genes have been reported. Here, we developed a novel defective-rescue BmNPV Bacmid (reBmBac) expression system. A CopyControl origin of replication was introduced into the viral genome to facilitate its genetic manipulation in Escherichia coli and to ensure the preparation of large amounts of high quality reBmBac DNA as well as high quality recombinant baculoviruses. The ORF1629, cathepsin and chitinase genes were partially deleted or rendered defective to improve the efficiency of recombinant baculovirus generation and the expression of foreign genes. The system was validated by the successful expression of luciferase reporter gene and porcine interferon γ. This system can be used to produce batches of recombinant baculoviruses and target proteins rapidly and efficiently in silkworms. PMID:27008267

  17. Double promoter expression systems for recombinant protein production by industrial microorganisms.

    PubMed

    Öztürk, Sibel; Ergün, Burcu Gündüz; Çalık, Pınar

    2017-09-12

    Using double promoter expression systems is a promising approach to increase heterologous protein production. In this review, current double promoter expression systems for the production of recombinant proteins (r-proteins) by industrially important bacteria, Bacillus subtilis and Escherichia coli; and yeasts, Saccharomyces cerevisiae and Pichia pastoris, are discussed by assessing their potentials and drawbacks. Double promoter expression systems need to be designed to maintain a higher specific product formation rate within the production domain. While bacterial double promoter systems have been constructed as chimeric tandem promoters, yeast dual promoter systems have been developed as separate expression cassettes. To increase production and productivity, the optimal transcriptional activity should be justified either by simultaneously satisfying the requirements of both promoters, or by consecutively stimulating the changeover from one to another in a biphasic process or via successive-iterations. Thus, considering the dynamics of a fermentation process, double promoters can be classified according to their operational mechanisms, as: i) consecutively operating double promoter systems, and ii) simultaneously operating double promoter systems. Among these metabolic design strategies, extending the expression period with two promoters activated under different conditions, or enhancing the transcriptional activity with two promoters activated under similar conditions within the production domain, can be applied independently from the host. Novel studies with new insights, which aim a rational systematic design and construction of dual promoter expression vectors with tailored transcriptional activity, will empower r-protein production with enhanced production and productivity. Finally, the current state-of-the-art review emphasizes the advantages of double promoter systems along with the necessity for discovering new promoters for the development of more

  18. Comparison of enterococci and coliform microorganisms in commercially produced pecan nut meats.

    PubMed

    HYNDMAN, J B

    1963-05-01

    Pecan nut meats in the unbroken shell are sterile for enteric microorganisms. Recovery of coliform microorganisms or enterococci from finished pecan nut meats indicated contact contamination, assuming the tempering procedures to be satisfactory. Results of specific studies, designed toward developing background data on the sanitary significance of enterococci and coliform microorganisms in the production of pecan meats are reported. Unbroken pecan nuts or nut meats from various stages of shelling operations were diluted with a phosphate-buffered diluent. Serial dilutions were inoculated into Lactose Broth and Azide Dextrose Broth. The lactose fermentors were carried through indole, methyl red, Voges-Proskauer, and citrate reactions; the positive Azide Dextrose cultures were confirmed in Ethyl Violet Azide Broth and microscopically. Viable plate counts were obtained. Enterococci were found resistant to many deterrent factors affecting coliforms. Recoveries of enterococci were detected long after pollution had occurred. Little correlation was found between enterococcal recovery and observed insanitary practices in commercial shelling operations. Using the coliaerogenes group and, specifically, Escherichia coli as a sanitation index, microorganisms allowed accurate appraisal of tempering, personnel practices, and contact surface contaminating factors. It is felt this was due, in part, to the more delicate growth characteristics of E. coli. The fact that other pathogenic microorganisms, capable of causing gastrointestinal upsets, are associated with the presence of E. coli introduces a health factor which is important to regulatory agencies concerned with consumer protection.

  19. Construction of a stable genetically engineered rhamnolipid-producing microorganism for remediation of pyrene-contaminated soil.

    PubMed

    Cao, Li; Wang, Qian; Zhang, Ji; Li, Chao; Yan, Xin; Lou, Xu; Xia, Yali; Hong, Qing; Li, Shunpeng

    2012-09-01

    One rhamnolipid-producing bacterial strain named Pseudomonas aeruginosa BSFD5 was isolated and characterized. Its rhlABRI cassette including necessary genes for rhamnolipid synthesis was cloned and transformed into the chromosome of P. putida KT2440 by a new random transposon vector without introducing antibiotic-resistance marker, generating a genetically engineered microorganism named P. putida KT2440-rhlABRI, which could stably express the rhlABRI cassette and produce rhamnolipid at a yield of 1.68 g l(-1). In experiments using natural soil, it was shown that P. putida KT2440-rhlABRI could increase the dissolution of pyrene and thus promote its degradation by indigenous microorganisms. P. putida KT2440-rhlABRI thus demonstrated potential for enhancing the remediation of soils contaminated with polycyclic aromatic hydrocarbons.

  20. Simple, effective protein extraction method and proteomics analysis from polyunsaturated fatty acids-producing micro-organisms.

    PubMed

    Ling, Xueping; Guo, Jing; Zheng, Chuqiang; Ye, Chiming; Lu, Yinghua; Pan, Xueshan; Chen, Zhengqi; Ng, I-Son

    2015-12-01

    Polyunsaturated fatty acids (PUFAs) are valuable ingredients in the food and pharmaceutical products due to their beneficial influence on human health. Most studies paid attention on the production of PUFAs from oleaginous micro-organisms but seldom on the comparative proteomics of cells. In the study, three methods (i.e., cold shock, acetone precipitation and ethanol precipitation) for lipid removal from crude protein extracts were applied in different PUFAs-producing micro-organisms. Among the selective strains, Schizochytrium was used as an oleaginous strain with high lipid of 60.3 (w/w%) in biomass. The Mortierella alpina and Cunninghamella echinulata were chosen as the low-lipid-content strains with 25.8 (w/w%) and 21.8 (w/w%) of lipid in biomass, respectively. The cold shock resulted as the most effective method for lipid removed, thus obtained higher protein amount for Schizochytrium. Moreover, from the comparative proteomics for the three PUFAs-producing strains, it showed more significant proteins of up or down-regulation were explored under cold shock treatment. Therefore, the essential proteins (i.e., polyunsaturated fatty acid synthase) and regulating proteins were observed. In conclusion, this study provides a valuable and practical approach for analysis of high PUFAs-producing strains at the proteomics level, and would further accelerate the understanding of the metabolic flux in oleaginous micro-organisms.

  1. Investigation of biosurfactant-producing indigenous microorganisms that enhance residue oil recovery in an oil reservoir after polymer flooding.

    PubMed

    She, Yue-Hui; Zhang, Fan; Xia, Jing-Jing; Kong, Shu-Qiong; Wang, Zheng-Liang; Shu, Fu-Chang; Hu, Ji-Ming

    2011-01-01

    Three biosurfactant-producing indigenous microorganisms (XDS1, XDS2, XDS3) were isolated from a petroleum reservoir in the Daqing Oilfield (China) after polymer flooding. Their metabolic, biochemical, and oil-degradation characteristics, as well as their oil displacement in the core were studied. These indigenous microorganisms were identified as short rod bacillus bacteria with white color, round shape, a protruding structure, and a rough surface. Strains have peritrichous flagella, are able to produce endospores, are sporangia, and are clearly swollen and terminal. Bacterial cultures show that the oil-spreading values of the fermentation fluid containing all three strains are more than 4.5 cm (diameter) with an approximate 25 mN/m surface tension. The hydrocarbon degradation rates of each of the three strains exceeded 50%, with the highest achieving 84%. Several oil recovery agents were produced following degradation. At the same time, the heavy components of crude oil were degraded into light components, and their flow characteristics were also improved. The surface tension and viscosity of the crude oil decreased after being treated by the three strains of microorganisms. The core-flooding tests showed that the incremental oil recoveries were 4.89-6.96%. Thus, XDS123 treatment may represent a viable method for microbial-enhanced oil recovery.

  2. Isolation and characterization of a novel thraustochytrid-like microorganism that efficiently produces docosahexaenoic acid.

    PubMed

    Perveen, Zakia; Ando, Hitomi; Ueno, Akio; Ito, Yukiya; Yamamoto, Yusuke; Yamada, Yohko; Takagi, Tomoko; Kaneko, Takako; Kogame, Kazuhiro; Okuyama, Hidetoshi

    2006-02-01

    A thraustochytrid-like microorganism (strain 12B) was isolated from the mangrove area of Okinawa, Japan. On the basis of its ectoplasmic net structure and biflagellate zoospores we determined strain 12B to be a novel member of the phylum Labyrinthulomycota in the kingdom Protoctista. When grown on glucose/seawater at 28 degrees C, it had a lipid content of 58% with docosahexaenoic acid (DHA; 22:6 n-3) at 43% of the total fatty acids. It had a growth rate of 0.38 h(-1). The DHA production rate of 2.8 +/- 0.7 g l(-1) day(-1) is the highest value reported for any microorganism.

  3. A rapid, efficient and sensitive plate assay for detection and screening of l-asparaginase-producing microorganisms.

    PubMed

    Mahajan, Richi V; Saran, Saurabh; Saxena, Rajendra K; Srivastava, Ayush K

    2013-04-01

    l-Asparaginase-producing microbes are conventionally screened on phenol red l-asparagine-containing plates. However, sometimes the contrast of the zone obtained (between yellow and pink) is not very sharp and distinct. In the present investigation, an improved method for screening of the microorganisms producing extracellular l-asparaginase is reported wherein bromothymol blue (BTB) is incorporated as pH indicator in l-asparagine-containing medium instead of phenol red. Plates containing BTB at acidic pH are yellow and turn dark blue at alkaline pH. Thus, a dense dark blue zone is formed around microbial colonies producing l-asparaginase, differentiating between enzyme producers and non-producers. The present method is more sensitive and accurate than the conventional method for screening of both fungi and bacteria producing extracellular l-asparaginase. Furthermore, BTB gives a transient green colour at neutral pH (7.0) and dark blue colour at higher pH 8.0-9.0, indicating the potency of the microorganism for l-asparaginase production.

  4. Generation of polyclonal antibodies against recombinant human glucocerebrosidase produced in Escherichia coli.

    PubMed

    Novo, Juliana Branco; Oliveira, Maria Leonor Sarno; Magalhães, Geraldo Santana; Morganti, Ligia; Raw, Isaías; Ho, Paulo Lee

    2010-11-01

    Deficiency of the lysosomal glucocerebrosidase (GCR) enzyme results in Gaucher's disease, the most common inherited storage disorder. Treatment consists of enzyme replacement therapy by the administration of recombinant GCR produced in Chinese hamster ovary cells. The production of anti-GCR antibodies has already been described with placenta-derived human GCR that requires successive chromatographic procedures. Here, we report a practical and efficient method to obtain anti-GCR polyclonal antibodies against recombinant GCR produced in Escherichia coli and further purified by a single step through nickel affinity chromatography. The purified GCR was used to immunize BALB/c mice and the induction of anti-GCR antibodies was evaluated by enzyme-linked immunosorbent assay. The specificity of the antiserum was also evaluated by western blot analysis against recombinant GCR produced by COS-7 cells or against endogenous GCR of human cell lines. GCR was strongly recognized by the produced antibodies, either as cell-associated or as secreted forms. The detected molecular masses of 59-66 kDa are in accordance to the expected size for glycosylated GCR. The GCR produced in E. coli would facilitate the production of polyclonal (shown here) and monoclonal antibodies and their use in the characterization of new biosimilar recombinant GCRs coming in the near future.

  5. Antibiotic producing microorganisms from River Wiwi, Lake Bosomtwe and the Gulf of Guinea at Doakor Sea Beach, Ghana.

    PubMed

    Tawiah, Adelaide A; Gbedema, Stephen Y; Adu, Francis; Boamah, Vivian E; Annan, Kofi

    2012-10-16

    Microorganisms have provided a wealth of metabolites with interesting activities such as antimicrobial, antiviral and anticancer. In this study, a total of 119 aquatic microbial isolates from 30 samples (taken from water bodies in Ghana) were screened by the agar-well diffusion method for ability to produce antibacterial-metabolites. Antibacterial activity was exhibited by 27 of the isolates (14 bacteria, 9 actinomycetes and 4 fungi) against at least one of the indicator microorganisms: Enterococcus faecalis (ATCC 29212), Bacillus thuringiensis (ATCC 13838), Pseudomonas aeruginosa (ATCC 27853), Staphylococcus aureus (ATCC 25923), Proteus vulgaris (NCTC 4635) and Bacillus Subtilis (NCTC 10073). A sea isolate MAI2 (identified as a strain of Pseudomonas aeruginosa) exhibited the highest antibacterial activity (lowest zone of inhibition = 22 mm). The metabolites of MAI2 extracted with chloroform were stable to heat and gave minimum inhibitory concentrations ranging between 250 and 2000 μg/ml. Bioautography of the extract revealed seven active components. This study has therefore uncovered the potential of water bodies in the West African sub-region as reservoirs of potent bioactive metabolite producing microorganisms.

  6. Antibiotic producing microorganisms from River Wiwi, Lake Bosomtwe and the Gulf of Guinea at Doakor Sea Beach, Ghana

    PubMed Central

    2012-01-01

    Background Microorganisms have provided a wealth of metabolites with interesting activities such as antimicrobial, antiviral and anticancer. In this study, a total of 119 aquatic microbial isolates from 30 samples (taken from water bodies in Ghana) were screened by the agar-well diffusion method for ability to produce antibacterial-metabolites. Results Antibacterial activity was exhibited by 27 of the isolates (14 bacteria, 9 actinomycetes and 4 fungi) against at least one of the indicator microorganisms: Enterococcus faecalis (ATCC 29212), Bacillus thuringiensis (ATCC 13838), Pseudomonas aeruginosa (ATCC 27853), Staphylococcus aureus (ATCC 25923), Proteus vulgaris (NCTC 4635) and Bacillus Subtilis (NCTC 10073). A sea isolate MAI2 (identified as a strain of Pseudomonas aeruginosa) exhibited the highest antibacterial activity (lowest zone of inhibition = 22 mm). The metabolites of MAI2 extracted with chloroform were stable to heat and gave minimum inhibitory concentrations ranging between 250 and 2000 μg/ml. Bioautography of the extract revealed seven active components. Conclusion This study has therefore uncovered the potential of water bodies in the West African sub-region as reservoirs of potent bioactive metabolite producing microorganisms. PMID:23072432

  7. Polymorphonuclear counts in ascitic fluid and microorganisms producing spontaneous bacterial peritonitis: an under-recognized relationship.

    PubMed

    Ariza, Xavier; Lora-Tamayo, Jaime; Castellote, José; Xiol, Xavier; Ariza, Javier

    2013-10-01

    BACKGROUND AND AIMS. In cirrhotic patients with spontaneous bacterial peritonitis (SBP) higher polymorphonuclear (PMN) count in ascitic fluid have been reported in infections caused by Gram-negative bacilli (GNB) as opposed to Gram-positive cocci (GPC). However, the influence of other associated factors on the PMN count, such as the specific microorganism causing the episode of SBP, has not been well established. METHODS. Retrospective observational study of 194 episodes of positive ascitic and/or blood culture SBP in 159 patients with liver cirrhosis (2001-2009). Parameters associated with PMN count in ascitic fluid at diagnosis were evaluated. RESULTS. The multivariate analysis (model 1) showed that a virulent etiology of the infection [coefficient 3.941 (95% confidence interval (95 CI): 0.421-7.461)] and the model for end-stage liver disease (MELD) score [coefficient 0.196 (95 CI: 0.007-0.384)] were positively associated with the PMN count in ascites, while a nosocomial acquisition was inversely associated [coefficient -3.546 (95 CI: -6.855 - -0.238)]. A nonsignificant trend toward higher PMN count was found in GNB versus GPC, but there were differences between groups of microorganisms: pyogenic streptococci [median (p25-p75): 3211 (1615-8004)], Enterobacteriaceae [2958 (917-7690)], Vibrionaceae [9215 (375-17280)], nonfermenting GNB [1384 (565-3865)], viridans group streptococci [1044 (503-2354)] and enterococci [1050 (476-4655)](p = 0.005). No clear cut-offs of ascitic PMN count predicting a particular etiology could be calculated out of these data. CONCLUSIONS. In cirrhotic patients with SBP, the causing microorganism, the place of acquisition of the infection and the host liver condition were the main factors determining PMN count in ascitic fluid. Third-generation cephalosporin resistance was associated with low PMN count probably because this group included bacteria with inherent low virulence.

  8. Detergent composition comprising a cellulase containing cell-free fermentate produced from microorganism ATCC 55702 or mutant thereof

    DOEpatents

    Dees, H. Craig

    1998-01-01

    Bacteria which produce large amounts of a cellulase-containing cell-free fermentate have been identified. The original bacterium (ATCC 55703) was genetically altered using nitrosoguanidine (MNNG) treatment to produce the enhanced cellulase producing bacterium (ATCC 55702), which was identified through replicate plating. ATCC 55702 has improved characteristics and qualities for the degradation of cellulosic waste materials for fuel production, food processing, textile processing, and other industrial applications. ATCC 55702 is an improved bacterial host for genetic manipulations using recombinant DNA techniques, and is less likely to destroy genetic manipulations using standard mutagenesis techniques.

  9. Detergent composition comprising a cellulase containing cell-free fermentate produced from microorganism ATCC 55702 or mutant thereof

    DOEpatents

    Dees, H.C.

    1998-07-14

    Bacteria which produce large amounts of a cellulase-containing cell-free fermentate have been identified. The original bacterium (ATCC 55703) was genetically altered using nitrosoguanidine (MNNG) treatment to produce the enhanced cellulase producing bacterium (ATCC 55702), which was identified through replicate plating. ATCC 55702 has improved characteristics and qualities for the degradation of cellulosic waste materials for fuel production, food processing, textile processing, and other industrial applications. ATCC 55702 is an improved bacterial host for genetic manipulations using recombinant DNA techniques, and is less likely to destroy genetic manipulations using standard mutagenesis techniques. 5 figs.

  10. Recent development of two chitinase inhibitors, Argifin and Argadin, produced by soil microorganisms

    PubMed Central

    Hirose, Tomoyasu; Sunazuka, Toshiaki; Ōmura, Satoshi

    2010-01-01

    Chitin, the second most abundant polysaccharide in nature, occurs in fungi, some algae and many invertebrates, including insects. Thus, chitin synthesis and degradation could represent specific targets for fungicides and insecticides. Chitinases hydrolyze chitin into oligomers of N-acetyl-d-glucosamine at key points in the life cycles of organisms, consequently, chitinase inhibitors have become subject of increasing interest. This review covers the development of two chitinase inhibitors of natural origin, Argifin and Argadin, isolated from the cultured broth of microorganisms in our laboratory. In particular, the practical total synthesis of these natural products, the synthesis of lead compounds via computer-aided rational molecular design, and discovery methods that generate only highly-active compounds using a kinetic target(chitinase)-guided synthesis approach (termed in situ click chemistry) are described. PMID:20154467

  11. Process for producing modified microorganisms for oil treatment at high temperatures, pressures and salinity

    DOEpatents

    Premuzic, Eugene T.; Lin, Mow

    1996-02-20

    This invention relates to the preparation of new, modified organisms, through challenge growth processes, that are viable in the extreme temperature, pressure and pH conditions and salt concentrations of an oil reservoir and that are suitable for use in microbial enhanced oil recovery. The modified microorganisms of the present invention are used to enhance oil recovery and remove sulfur compounds and metals from the crude oil. The processes are comprised of steps which successively limit the carbon sources and increase the temperature, pressure and salinity of the media. This is done until microbial strains are obtained that are capable of growing in essentially crude oil as a carbon source and at a temperature range from about 70.degree. C. to 90.degree. C., at a pressure range from about 2,000 to 2,500 psi and at a salinity range from about 1.3 to 35%.

  12. Process for producing modified microorganisms for oil treatment at high temperatures, pressures and salinity

    DOEpatents

    Premuzic, E.T.; Lin, M.

    1996-02-20

    This invention relates to the preparation of new, modified organisms, through challenge growth processes, that are viable in the extreme temperature, pressure and pH conditions and salt concentrations of an oil reservoir and that are suitable for use in microbial enhanced oil recovery. The modified microorganisms of the present invention are used to enhance oil recovery and remove sulfur compounds and metals from the crude oil. The processes are comprised of steps which successively limit the carbon sources and increase the temperature, pressure and salinity of the media. This is done until microbial strains are obtained that are capable of growing in essentially crude oil as a carbon source and at a temperature range from about 70 C to 90 C, at a pressure range from about 2,000 to 2,500 psi and at a salinity range from about 1.3 to 35%. 68 figs.

  13. Protein folding and conformational stress in microbial cells producing recombinant proteins: a host comparative overview

    PubMed Central

    Gasser, Brigitte; Saloheimo, Markku; Rinas, Ursula; Dragosits, Martin; Rodríguez-Carmona, Escarlata; Baumann, Kristin; Giuliani, Maria; Parrilli, Ermenegilda; Branduardi, Paola; Lang, Christine; Porro, Danilo; Ferrer, Pau; Tutino, Maria Luisa; Mattanovich, Diethard; Villaverde, Antonio

    2008-01-01

    Different species of microorganisms including yeasts, filamentous fungi and bacteria have been used in the past 25 years for the controlled production of foreign proteins of scientific, pharmacological or industrial interest. A major obstacle for protein production processes and a limit to overall success has been the abundance of misfolded polypeptides, which fail to reach their native conformation. The presence of misfolded or folding-reluctant protein species causes considerable stress in host cells. The characterization of such adverse conditions and the elicited cell responses have permitted to better understand the physiology and molecular biology of conformational stress. Therefore, microbial cell factories for recombinant protein production are depicted here as a source of knowledge that has considerably helped to picture the extremely rich landscape of in vivo protein folding, and the main cellular players of this complex process are described for the most important cell factories used for biotechnological purposes. PMID:18394160

  14. An emerging public health problem: acquired carbapenemase-producing microorganisms are present in food-producing animals, their environment, companion animals and wild birds.

    PubMed

    Guerra, Beatriz; Fischer, Jennie; Helmuth, Reiner

    2014-07-16

    Worldwide, the emergence and global spread of microorganisms with acquired carbapenemases is of great concern. The reservoirs for such organisms are increasing, not only in hospitals, but also in the community and environment. A new and important development is the presence of such organisms in livestock, companion animals and wildlife. During the last three years, carbapenemase-producing Escherichia coli, Salmonella spp. (VIM-1 producers) and Acinetobacter spp. (producing OXA-23 and NDM-1) in livestock animals (poultry, cattle and swine) and their environment have been reported. In addition, the isolation of NDM-1-producing E. coli, OXA-48 in E. coli and Klebsiella pneumoniae or OXA-23 in Acinetobacter spp. from companion animals (cats, dogs or horses) has also been observed. Other reports have described the presence of NDM-1-producing Salmonella isolated from wild birds, as well as OXA-23-like-producing Acinetobacter baumannii in ectoparasites. However, until now carbapenemase producers from foods have not been detected. For humans in contrast carbapenem-producing Salmonella isolates are increasingly reported. The real prevalence of carbapenemase-encoding genes in zoonotic bacteria or commensals from animals is unknown. Consequently, there is a need for intensified surveillance on the occurrence of carbapenemase-producing bacteria in the food chain and other animal sources in order to assist in the formulation of measures to prevent their potential spread. Copyright © 2014 Elsevier B.V. All rights reserved.

  15. Producing recombinant therapeutic glycoproteins with enhanced sialylation using CHO-gmt4 glycosylation mutant cells

    PubMed Central

    Goh, John SY; Liu, Yingwei; Chan, Kah Fai; Wan, Corrine; Teo, Gavin; Zhang, Peiqing; Zhang, Yuanxing; Song, Zhiwei

    2014-01-01

    Recombinant glycoprotein drugs require proper glycosylation for optimal therapeutic efficacy. Glycoprotein therapeutics are rapidly removed from circulation and have reduced efficacy if they are poorly sialylated. Ricinus communis agglutinin-I (RCA-I) was found highly toxic to wild-type CHO-K1 cells and all the mutants that survived RCA-I treatment contained a dysfunctional N-acetylglucosaminyltransferase I (GnT I) gene. These mutants are named CHO-gmt4 cells. Interestingly, upon restoration of GnT I, the sialylation of a model glycoprotein, erythropoietin, produced in CHO-gmt4 cells was shown to be superior to that produced in wild-type CHO-K1 cells. This addendum summarizes the applicability of this cell line, from transient to stable expression of the recombinant protein, and from a lab scale to an industrial scale perfusion bioreactor. In addition, CHO-gmt4 cells can be used to produce glycoproteins with mannose-terminated N-glycans. Recombinant glucocerebrosidase produced by CHO-gmt4 cells will not require glycan remodeling and may be directly used to treat patients with Gaucher disease. CHO-gmt4 cells can also be used to produce other glycoprotein therapeutics which target cells expressing mannose receptors. PMID:24911584

  16. Producing recombinant therapeutic glycoproteins with enhanced sialylation using CHO-gmt4 glycosylation mutant cells.

    PubMed

    Goh, John S Y; Liu, Yingwei; Chan, Kah Fai; Wan, Corrine; Teo, Gavin; Zhang, Peiqing; Zhang, Yuanxing; Song, Zhiwei

    2014-01-01

    Recombinant glycoprotein drugs require proper glycosylation for optimal therapeutic efficacy. Glycoprotein therapeutics are rapidly removed from circulation and have reduced efficacy if they are poorly sialylated. Ricinus communis agglutinin-I (RCA-I) was found highly toxic to wild-type CHO-K1 cells and all the mutants that survived RCA-I treatment contained a dysfunctional N-acetylglucosaminyltransferase I (GnT I) gene. These mutants are named CHO-gmt4 cells. Interestingly, upon restoration of GnT I, the sialylation of a model glycoprotein, erythropoietin, produced in CHO-gmt4 cells was shown to be superior to that produced in wild-type CHO-K1 cells. This addendum summarizes the applicability of this cell line, from transient to stable expression of the recombinant protein, and from a lab scale to an industrial scale perfusion bioreactor. In addition, CHO-gmt4 cells can be used to produce glycoproteins with mannose-terminated N-glycans. Recombinant glucocerebrosidase produced by CHO-gmt4 cells will not require glycan remodeling and may be directly used to treat patients with Gaucher disease. CHO-gmt4 cells can also be used to produce other glycoprotein therapeutics which target cells expressing mannose receptors.

  17. Ethanol production from paper sludge by simultaneous saccharification and co-fermentation using recombinant xylose-fermenting microorganisms.

    PubMed

    Zhang, Jiayi; Lynd, Lee R

    2010-10-01

    Simultaneous saccharification and co-fermentation (SSCF) of waste paper sludge to ethanol was investigated using two recombinant xylose-fermenting microbes: Zymomonas mobilis 8b and Saccharomyces cerevisiae RWB222. S. cerevisiae RWB222 produced over 40 g/L ethanol with a yield of 0.39 g ethanol/g carbohydrate on paper sludge at 37 degrees C, while similar titers and yields were achieved by Z. mobilis 8b at 30 degrees C. Both S. cerevisiae RWB222 and Z. mobilis 8b exhibited decreasing cell viability at 37 degrees C when producing over 40 g/L ethanol. A high ethanol concentration can account for S. cerevisiae RWB222 viability loss, but ethanol concentration was not the only factor influencing Z. mobilis 8b viability loss at 37 degrees C. Over 3 g/L residual glucose was observed at the end of paper sludge SSCF by Z. mobilis 8b, and a statistical analysis revealed that a high calcium concentration originating from paper sludge, a high ethanol concentration, and a high temperature were the key interactive factors resulting in glucose accumulation. The highest ethanol yields were achieved by SSCF of paper sludge with S. cerevisiae RWB222 at 37 degrees C and Z. mobilis 8b at 30 degrees C. With good sugar consumption at 37 degrees C, S. cerevisiae RWB222 was able to gain an improvement in the polysaccharide to sugar yield compared to that at 30 degrees C, whereas Z. mobilis 8b at 30 degrees C had a lower polysaccharide to sugar yield, but a higher sugar to ethanol yield than S. cerevisiae. Both organisms under optimal conditions achieved a 19% higher overall conversion of paper sludge to ethanol than the non-xylose utilizing S. cerevisiae D5A at its optimal process temperature of 37 degrees C.

  18. Activity and characterization of secondary metabolites produced by a new microorganism for control of plant diseases.

    PubMed

    Ko, Wen-Hsiung; Tsou, Yi-Jung; Lin, Mei-Ju; Chern, Lih-Ling

    2010-09-30

    Microorganisms capable of utilizing vegetable tissues for growth in soils were isolated and their vegetable broth cultures were individually sprayed directly on leaves to test their ability to control Phytophthora blight of bell pepper caused by Phytophthora capsici. Liquid culture of Streptomyces strain TKA-5, a previously undescribed species obtained in this study, displayed several desirable disease control characteristics in nature, including high potency, long lasting and ability to control also black leaf spot of spoon cabbage caused by Alternaria brassicicolca. The extract was fungicidal to P. capsici but fungistatic to A. brassicicola. It was stable at high temperature and high pH. However, after exposure to pH 2 for 24h, the extract was no longer inhibitory to P. capsici although it was still strongly inhibitory to A. brassicicola. After treatment with cation or anion exchange resins, the extract lost its inhibitory effect against P. capsici but not A. brassicicola. The results suggest that the extract contained two different kinds of inhibitory metabolites, one against P. capsici with both positive and negative charges on its molecule and another against A. brassicicola with no charges on its molecule. The inhibitory metabolites were soluble in ethanol or methanol but not in water, ether or chloroform. They were dialyzable in the membrane tubing with molecular weight cut-off of 10,000, 1000 or 500 but not 100, indicating that the inhibitors have a molecular weight between 500 and 100. Results also showed that both inhibitors are not proteins.

  19. Recombinant human N-acetylgalactosamine-6-sulfate sulfatase (GALNS) produced in the methylotrophic yeast Pichia pastoris

    PubMed Central

    Rodríguez-López, Alexander; Alméciga-Díaz, Carlos J.; Sánchez, Jhonnathan; Moreno, Jefferson; Beltran, Laura; Díaz, Dennis; Pardo, Andrea; Ramírez, Aura María; Espejo-Mojica, Angela J.; Pimentel, Luisa; Barrera, Luis A.

    2016-01-01

    Mucopolysaccharidosis IV A (MPS IV A, Morquio A disease) is a lysosomal storage disease (LSD) produced by mutations on N-acetylgalactosamine-6-sulfate sulfatase (GALNS). Recently an enzyme replacement therapy (ERT) for this disease was approved using a recombinant enzyme produced in CHO cells. Previously, we reported the production of an active GALNS enzyme in Escherichia coli that showed similar stability properties to that of a recombinant mammalian enzyme though it was not taken-up by culture cells. In this study, we showed the production of the human recombinant GALNS in the methylotrophic yeast Pichia pastoris GS115 (prGALNS). We observed that removal of native signal peptide and co-expression with human formylglycine-generating enzyme (SUMF1) allowed an improvement of 4.5-fold in the specific GALNS activity. prGALNS enzyme showed a high stability at 4 °C, while the activity was markedly reduced at 37 and 45 °C. It was noteworthy that prGALNS was taken-up by HEK293 cells and human skin fibroblasts in a dose-dependent manner through a process potentially mediated by an endocytic pathway, without any additional protein or host modification. The results show the potential of P. pastoris in the production of a human recombinant GALNS for the development of an ERT for Morquio A. PMID:27378276

  20. The effects of rare earth elements on an anaerobic hydrogen producing microorganism

    NASA Astrophysics Data System (ADS)

    Fujita, Y.; St Jeor, J. D.; Reed, D. W.

    2016-12-01

    Rapid growth of new energy technologies and consumer electronics is leading to increased fluxes of rare earth elements (REE), during the phases of resource extraction, product usage, recycling, and disposal. However, little is known about the impacts of these increased REE fluxes on environmental ecosystems, whether natural or engineered (e.g., biological waste treatment systems). We have been evaluating the effects of europium and yttrium on hydrogen production by an anaerobic fermenting microorganism, Sporacetigenium mesophilum, originally isolated from an anaerobic digester at a wastewater treatment plant.1 Europium and yttrium are important components of phosphors used in fluorescent lighting, and are expected to be recycled in larger quantities in the future. Also tested was the compound tributyl phosphate (TBP), a widely used complexing agent in lanthanide and actinide separations. TBP and related compounds may be used in recycling processes for REE. S. mesophilumcultures were amended with Eu at 100 ppb, 1 ppm and 10 ppm and hydrogen production was measured. While the lowest Eu concentration had minimal effect on hydrogen production compared to the no Eu control, the two higher Eu amendment levels appeared to enhance hydrogen production. TBP at 0.1 g/L completely inhibited hydrogen production. Measurements of aqueous Eu concentrations indicated that >85% of the added Eu remained soluble at all three of the Eu addition levels tested. Experiments to ascertain whether enhancement (or inhibition) occurs at even higher Eu concentrations are underway, as are corresponding experiments with yttrium. This work contributes to the assessment of the potential impacts of increased REE recycling and processing on ecosystems, and supports decision making with respect to disposal of wastewaters generated during these industrial practices. 1Chen, S., Song, L. and X. Dong. Int J. Syst. Evol. Microbiol. 56, 721-725, doi: 10.1099/ijs.0.63686-0 (2006).

  1. Using the second law of thermodynamics for enrichment and isolation of microorganisms to produce fuel alcohols or hydrocarbons.

    PubMed

    Kohn, Richard A; Kim, Seon-Woo

    2015-10-07

    Fermentation of crops, waste biomass, or gases has been proposed as a means to produce desired chemicals and renewable fuels. The second law of thermodynamics has been shown to determine the net direction of metabolite flow in fermentation processes. In this article, we describe a process to isolate and direct the evolution of microorganisms that convert cellulosic biomass or gaseous CO2 and H2 to biofuels such as ethanol, 1-butanol, butane, or hexane (among others). Mathematical models of fermentation elucidated sets of conditions that thermodynamically favor synthesis of desired products. When these conditions were applied to mixed cultures from the rumen of a cow, bacteria that produced alcohols or alkanes were isolated. The examples demonstrate the first use of thermodynamic analysis to isolate bacteria and control fermentation processes for biofuel production among other uses.

  2. New detection method for hydrogen gas for screening hydrogen-producing microorganisms using water-soluble wilkinson's catalyst derivative.

    PubMed

    Katsuda, Tomohisa; Ooshima, Hiroshi; Azuma, Masayuki; Kato, Jyoji

    2006-09-01

    A water-soluble color indicator was developed for the effective screening of hydrogen-producing microorganisms. This indicator consists of a coloring agent and a water-soluble derivative of Wilkinson's catalyst. Wilkinson's catalyst, Tris(triphenylphosphine) rhodium chloride, had been developed as a catalyst for the hydrogenation of olefins. We used a sulfonate of the catalyst for the hydrogenation of coloring agent in an aqueous medium. Several coloring agents, such as methyl orange, methyl red sodium, neutral red and Evan's blue, dissolved in water together with the sulfonated catalyst showed a change in color when hydrogen gas was fed into the solution by sparging at room temperature. We confirmed that methyl orange was decolorized by biologically produced hydrogen, when the photosynthetic bacterial strain Rhodobacter capsulatus ST-410 was grown in a medium containing 0.6 mM catalyst and 0.075 mM methyl orange in test tubes of 5 ml working volume.

  3. Overview of the purification of recombinant proteins produced in Escherichia coli.

    PubMed

    Wingfield, Paul T

    2003-02-01

    The updated version of this unit presents an overview of recombinant protein purification with special emphasis on proteins expressed in E. coli. The first section deals with information pertinent to protein purification that can be derived from translation of the cDNA sequence. This is followed by a discussion of common problems associated with bacterial protein expression. A flow chart summarizes approaches for establishing solubility and localization of bacterially produced proteins. Purification strategies for both soluble and insoluble proteins are also reviewed. A section on glycoproteins produced in bacteria in the nonglycosylated state is included to emphasize that, although they may not be useful for in vivo studies, such proteins are well suited for structural studies. Finally, protein handling, scale and aims of purification, and specialized equipment needed for recombinant protein purification and characterization are discussed. The methodologies and approaches described here are essentially suitable for laboratory-scale operations.

  4. [An effective scheme to produce recombinant uracil-DNA glycosylase of Escherichia coli for PCR diagnostics].

    PubMed

    Dmitrochenko, A E; Turiianskaia, O M; Gilep, A A; Usanov, S A; Iantsevich, A V

    2014-01-01

    An effective scheme has been developed to produce recombinant uracil-DNA glycosylase of Escherichia coli K12 intended to be used for PCR diagnostics, making it possible to achieve a high yield of the end product using a two-stage purification. The gene encoding this enzyme was cloned into the pCWori vector within the same reading frame with six residues of histidine in the C-erminal sequence. Using this vector and the E. coli DH5alpha, a host-vector expression system has been developed and conditions for protein synthesis have been optimized. To purify the protein, metal affinity chromatography with further dialysis was used to remove imidazole. The enzyme yield was no less than 60 mg of the end protein per 1 L of the culture medium. The concordance between amino acid sequences of the recombinant and native enzymes was proved by peptide mass fingerprinting and mass spectrometry. A rapid test to determine the activity of the enzyme preparation was suggested. It was found that the activity of 1.0 mg of the recombinant protein is no less than 3 x 10(3) units. The recombinant enzyme was most stable at pH 8.0 and an ionic strength of the solution equal to 200 mM; it lost its activity completely for 10 min at 60 degrees C. Storage during 1 h at 20 degrees C resulted in the loss of no more than 30% of activity. In the enzyme preparation, the activity of DNase was absent. The free energy of the unfolding of the protein globule of the recombinant uracil-DNA glycosylase is 23.1 +/- 0.2 kJ/mol. The data obtained indicate that the recombinant enzyme may be recommended for use in PCR diagnostics to prevent the appearance of false positive results caused by pollution of the reaction mixture by products of the preceding reactions.

  5. Antimicrobial activity of two South African honeys produced from indigenous Leucospermum cordifolium and Erica species on selected micro-organisms

    PubMed Central

    Basson, Nicolaas J; Grobler, Sias R

    2008-01-01

    Background Honey has been shown to have wound healing properties which can be ascribed to its antimicrobial activity. The antimicrobial activity can be effective against a broad spectrum of bacterial species especially those of medical importance. It has also been shown that there is considerable variation in the antimicrobial potency of different types of honey, which is impossible to predict. With this in mind we tested the antimicrobial activity of honeys produced from plants grown in South Africa for their antibacterial properties on selected standard strains of oral micro-organisms. Methods The honeys used were produced from the blossoms of Eucalyptus cladocalyx (Bluegum) trees, an indigenous South African plant Leucospermum cordifolium (Pincushion), a mixture of wild heather shrubs, mainly Erica species (Fynbos) and a Leptospermum scoparium (Manuka) honey. Only pure honey which had not been heated was used. The honeys were tested for their antimicrobial properties with a broth dilution method. Results Although the honeys produced some inhibitory effect on the growth of the micro-organisms, no exceptionally high activity occurred in the South African honeys. The carbohydrate concentration plays a key role in the antimicrobial activity of the honeys above 25%. However, these honeys do contain other antimicrobial properties that are effective against certain bacterial species at concentrations well below the hypertonic sugar concentration. The yeast C. albicans was more resistant to the honeys than the bacteria. The species S. anginosus and S. oralis were more sensitive to the honeys than the other test bacteria. Conclusion The honeys produced from indigenous wild flowers from South Africa had no exceptionally high activity that could afford medical grade status. PMID:18627601

  6. Ecology and characterization of polyhydroxyalkanoate-producing microorganisms on and in plants.

    PubMed

    Gasser, Ilona; Müller, Henry; Berg, Gabriele

    2009-10-01

    Polyhydroxyalkanoates are energy reserve polymers produced by bacteria to survive periods of starvation in natural habitats. Little is known about the ecology of polyhydroxyalkanoate-producing bacteria. To analyse the occurrence of this specific group on/in seven different plant species, a combined strategy containing culture-dependent and -independent methods was applied. Using microbial fingerprint techniques (single-strand conformation polymorphism analysis with specific primers for phaC gene encoding the key enzyme of the polyhydroxyalkanoate synthesis), a high number of bands were especially found for the rhizosphere. Furthermore, cluster analysis revealed plant species-specific communities. Isolation of bacteria, recognition of brightly refractile cytoplasmatic inclusions, lipophilic stainings and a PCR strategy targeted on the phaC gene were used as a culture-dependent strategy for the detection of polyhydroxyalkanoate-producing bacteria. Results again represent a high degree of plant specificity: the rhizosphere of sugar beet contained the highest number of positive strains. This was confirmed by quantitative PCR: the relative copy number of phaC was statistically and significantly enhanced in all rhizospheres in comparison with bulk soil. New polyhydroxyalkanoate-producing bacterial species were detected: for example, Burkholderia terricola, Lysobacter gummosus, Pseudomonas extremaustralis, Pseudomonas brassicacearum and Pseudomonas orientalis. Our results confirm the hypothesis that the rhizosphere is an interesting hidden reservoir for polyhydroxyalkanoate producers.

  7. Influence of Space-Flight Factors on the Properties of Microorganisms, Producers of Biologically Active Substances

    NASA Astrophysics Data System (ADS)

    Krasheninnikova, T. K.; Kanaeva, E. N.; Ukraintsev, A. D.; Smolyanaya, G. L.; Kuznetsov, N. V.; Panasyuk, M. I.; Shcherbakov, G. Ya.

    2001-07-01

    The following substances were isolated under the influence of space-flight factors in cosmic experiments aboard the Mirorbital station: an MIB-90 monoisolant, which is distinguished by its morphological and biochemical properties and enhanced productivity, was isolated from the Bacillus thuringiensis ssp. Kurstaki var. Z-52culture, which is a producer of the plant protection agent Lepidocide; and MIA-74 and MIP-89 monoisolants, which are highly active toward heavy petroleum fractions (C23 C33), were isolated from the Arthrobacter OC-1culture, which is a producer of biodegradants for petroleum.

  8. A gene responsible for prolyl-hydroxylation of moss-produced recombinant human erythropoietin

    PubMed Central

    Parsons, Juliana; Altmann, Friedrich; Graf, Manuela; Stadlmann, Johannes; Reski, Ralf; Decker, Eva L.

    2013-01-01

    Recombinant production of pharmaceutical proteins is crucial, not only for personalized medicine. While most biopharmaceuticals are currently produced in mammalian cell culture, plant-made pharmaceuticals gain momentum. Post-translational modifications in plants are similar to those in humans, however, existing differences may affect quality, safety and efficacy of the products. A frequent modification in higher eukaryotes is prolyl-4-hydroxylase (P4H)-catalysed prolyl-hydroxylation. P4H sequence recognition sites on target proteins differ between humans and plants leading to non-human posttranslational modifications of recombinant human proteins produced in plants. The resulting hydroxyprolines display the anchor for plant-specific O-glycosylation, which bears immunogenic potential for patients. Here we describe the identification of a plant gene responsible for non-human prolyl-hydroxylation of human erythropoietin (hEPO) recombinantly produced in plant (moss) bioreactors. Targeted ablation of this gene abolished undesired prolyl-hydroxylation of hEPO and thus paves the way for plant-made pharmaceuticals humanized via glyco-engineering in moss bioreactors. PMID:24145658

  9. A gene responsible for prolyl-hydroxylation of moss-produced recombinant human erythropoietin.

    PubMed

    Parsons, Juliana; Altmann, Friedrich; Graf, Manuela; Stadlmann, Johannes; Reski, Ralf; Decker, Eva L

    2013-10-22

    Recombinant production of pharmaceutical proteins is crucial, not only for personalized medicine. While most biopharmaceuticals are currently produced in mammalian cell culture, plant-made pharmaceuticals gain momentum. Post-translational modifications in plants are similar to those in humans, however, existing differences may affect quality, safety and efficacy of the products. A frequent modification in higher eukaryotes is prolyl-4-hydroxylase (P4H)-catalysed prolyl-hydroxylation. P4H sequence recognition sites on target proteins differ between humans and plants leading to non-human posttranslational modifications of recombinant human proteins produced in plants. The resulting hydroxyprolines display the anchor for plant-specific O-glycosylation, which bears immunogenic potential for patients. Here we describe the identification of a plant gene responsible for non-human prolyl-hydroxylation of human erythropoietin (hEPO) recombinantly produced in plant (moss) bioreactors. Targeted ablation of this gene abolished undesired prolyl-hydroxylation of hEPO and thus paves the way for plant-made pharmaceuticals humanized via glyco-engineering in moss bioreactors.

  10. Irrigation waters as a source of pathogenic microorganisms in produce: a review

    USDA-ARS?s Scientific Manuscript database

    There is increasing evidence that consumption of raw fresh produce is a major factor contributing to human gastrointestinal illness. A wide variety of pathogens contribute to food-borne illnesses, including bacteria (e.g., Salmonella, pathogenic E. coli), protozoa (e.g., Cryptosporidium, Giardia), ...

  11. Recombinant GDNF: Tetanus toxin fragment C fusion protein produced from insect cells

    SciTech Connect

    Li, Jianhong; Chian, Ru-Ju; Ay, Ilknur; Celia, Samuel A.; Kashi, Brenda B.; Tamrazian, Eric; Matthews, Jonathan C.; Remington, Mary P.; Pepinsky, R. Blake; Fishman, Paul S.; Brown, Robert H.; Francis, Jonathan W.

    2009-07-31

    Glial cell line-derived neurotrophic factor (GDNF) has potent survival-promoting effects on CNS motor neurons in experimental animals. Its therapeutic efficacy in humans, however, may have been limited by poor bioavailability to the brain and spinal cord. With a view toward improving delivery of GDNF to CNS motor neurons in vivo, we generated a recombinant fusion protein comprised of rat GDNF linked to the non-toxic, neuron-binding fragment of tetanus toxin. Recombinant GDNF:TTC produced from insect cells was a soluble homodimer like wild-type GDNF and was bi-functional with respect to GDNF and TTC activity. Like recombinant rat GDNF, the fusion protein increased levels of immunoreactive phosphoAkt in treated NB41A3-hGFR{alpha}-1 neuroblastoma cells. Like TTC, GDNF:TTC bound to immobilized ganglioside GT1b in vitro with high affinity and selectivity. These results support further testing of recombinant GDNF:TTC as a non-viral vector to improve delivery of GDNF to brain and spinal cord in vivo.

  12. Electrically conductive bacterial nanowires produced by Shewanella oneidensis strain MR-1 and other microorganisms.

    PubMed

    Gorby, Yuri A; Yanina, Svetlana; McLean, Jeffrey S; Rosso, Kevin M; Moyles, Dianne; Dohnalkova, Alice; Beveridge, Terry J; Chang, In Seop; Kim, Byung Hong; Kim, Kyung Shik; Culley, David E; Reed, Samantha B; Romine, Margaret F; Saffarini, Daad A; Hill, Eric A; Shi, Liang; Elias, Dwayne A; Kennedy, David W; Pinchuk, Grigoriy; Watanabe, Kazuya; Ishii, Shun'ichi; Logan, Bruce; Nealson, Kenneth H; Fredrickson, Jim K

    2006-07-25

    Shewanella oneidensis MR-1 produced electrically conductive pilus-like appendages called bacterial nanowires in direct response to electron-acceptor limitation. Mutants deficient in genes for c-type decaheme cytochromes MtrC and OmcA, and those that lacked a functional Type II secretion pathway displayed nanowires that were poorly conductive. These mutants were also deficient in their ability to reduce hydrous ferric oxide and in their ability to generate current in a microbial fuel cell. Nanowires produced by the oxygenic phototrophic cyanobacterium Synechocystis PCC6803 and the thermophilic, fermentative bacterium Pelotomaculum thermopropionicum reveal that electrically conductive appendages are not exclusive to dissimilatory metal-reducing bacteria and may, in fact, represent a common bacterial strategy for efficient electron transfer and energy distribution.

  13. Electrically conductive bacterial nanowires produced by Shewanella oneidensis strain MR-1 and other microorganisms

    PubMed Central

    Gorby, Yuri A.; Yanina, Svetlana; McLean, Jeffrey S.; Rosso, Kevin M.; Moyles, Dianne; Dohnalkova, Alice; Beveridge, Terry J.; Chang, In Seop; Kim, Byung Hong; Kim, Kyung Shik; Culley, David E.; Reed, Samantha B.; Romine, Margaret F.; Saffarini, Daad A.; Hill, Eric A.; Shi, Liang; Elias, Dwayne A.; Kennedy, David W.; Pinchuk, Grigoriy; Watanabe, Kazuya; Ishii, Shun’ichi; Logan, Bruce; Nealson, Kenneth H.; Fredrickson, Jim K.

    2006-01-01

    Shewanella oneidensis MR-1 produced electrically conductive pilus-like appendages called bacterial nanowires in direct response to electron-acceptor limitation. Mutants deficient in genes for c-type decaheme cytochromes MtrC and OmcA, and those that lacked a functional Type II secretion pathway displayed nanowires that were poorly conductive. These mutants were also deficient in their ability to reduce hydrous ferric oxide and in their ability to generate current in a microbial fuel cell. Nanowires produced by the oxygenic phototrophic cyanobacterium Synechocystis PCC6803 and the thermophilic, fermentative bacterium Pelotomaculum thermopropionicum reveal that electrically conductive appendages are not exclusive to dissimilatory metal-reducing bacteria and may, in fact, represent a common bacterial strategy for efficient electron transfer and energy distribution. PMID:16849424

  14. High-resolution gas chromatographic profiles of volatile organic compounds produced by microorganisms at refrigerated temperatures.

    PubMed Central

    Lee, M L; Smith, D L; Freeman, L R

    1979-01-01

    Three different strains of bacteria isolated from spoiled, uncooked chicken were grown in pure culture on Trypticase soy agar supplemented with yeast extract. The volatile organic compounds produced by each culture were concentrated on a porous polymer precolumn and analyzed by high-resolution gas chromatographic mass spectrometry. Twenty different compounds were identified. Both qualitative and quantitative differences in the chromatographic profiles from each culture were found. PMID:104660

  15. Biosurfactant-producing microorganism Pseudomonas sp. SB assists the phytoremediation of DDT-contaminated soil by two grass species.

    PubMed

    Wang, Beibei; Wang, Qingling; Liu, Wuxing; Liu, Xiaoyan; Hou, Jinyu; Teng, Ying; Luo, Yongming; Christie, Peter

    2017-09-01

    Phytoremediation together with microorganisms may confer the advantages of both phytoremediation and microbial remediation of soils containing organic contaminants. In this system biosurfactants produced by Pseudomonas sp. SB may effectively help to increase the bioavailability of organic pollutants and thereby enhance their microbial degradation in soil. Plants may enhance the rhizosphere environment for microorganisms and thus promote the bioremediation of contaminants. In the present pot experiment study, dichlorodiphenyltrichloroethane (DDT) residues underwent an apparent decline after soil bioremediation compared with the original soil. The removal efficiency of fertilizer + tall fescue, fertilizer + tall fescue + Pseudomonas, fertilizer + perennial ryegrass, and fertilizer + perennial ryegrass + Pseudomonas treatments were 59.4, 65.6, 69.0, and 65.9%, respectively, and were generally higher than that in the fertilizer control (40.3%). Principal coordinates analysis (PCoA) verifies that plant species greatly affected the soil bacterial community irrespective of inoculation with Pseudomonas sp. SB. Furthermore, community composition analysis shows that Proteobacteria, Acidobacteria and Actinobacteria were the three dominant phyla in all groups. In particular, the relative abundance of Pseudomonas for fertilizer + tall fescue + Pseudomonas (0.25%) was significantly greater than fertilizer + tall fescue and this was related to the DDT removal efficiency. Copyright © 2017 Elsevier Ltd. All rights reserved.

  16. Towards the molecular characterization of the stable producer phenotype of recombinant antibody-producing NS0 myeloma cells.

    PubMed

    Prieto, Y; Rojas, L; Hinojosa, L; González, I; Aguiar, D; de la Luz, K; Castillo, A; Pérez, R

    2011-08-01

    The loss of heterologous protein expression is one of the major problems faced by industrial cell line developers and has been reported by several authors. Therefore, the understanding of the mechanisms involved in the generation of stable and high producer cell lines is a critical issue, especially for those processes based on long term continuous cultures. We characterized two recombinant NS0 myeloma cell lines expressing Nimotuzumab, a humanized anti-human epidermal growth factor receptor (EGFR) antibody. The hR3/H7 clone is a stable producer obtained from the unstable hR3/t16 clone. The unstable clone was characterized by a bimodal distribution of intracellular immunoglobulin staining using flow cytometry. Loss of antibody production was due to the emergence of a non-producer cell subpopulation that increased with cell generation number. Immunoglobulin heavy chain (HC) and light chain (LC) ratio (HC/LC) was lower for the unstable phenotype. Proteomic maps using two dimensional gel electrophoresis (2DE) were obtained for both clones, at initial cell culture time and after 40 generations. Fifteen proteins potentially associated with the phenomenon of production stability were identified. The hR3/H7 stable clone showed an up-regulated expression pattern for most of these proteins. The regulation of recombinant antibody production by the host NS0 myeloma cell line most likely involves simultaneously cellular processes such as DNA transcription, mRNA processing, protein synthesis and folding, vesicular transport, glycolysis and energy production, according to the proteins identified in the present proteomic study.

  17. The Paleobiosphere: a novel device for the in vivo testing of hydrocarbon producing-utilizing microorganisms.

    PubMed

    Strobel, Gary; Booth, Eric; Schaible, George; Mends, Morgan Tess; Sears, Joe; Geary, Brad

    2013-04-01

    The construction and testing of a unique instrument, the Paleobiosphere, which mimics some of the conditions of the ancient earth, is described. The instrument provides an experimental testing system for determining if certain microbes, when provided an adequate environment, can degrade biological materials to produce fuel-like hydrocarbons in a relatively short time frame that become trapped by the shale. The conditions selected for testing included a particulate Montana shale (serving as the "Trap Shale"), plant materials (leaves and stems of three extant species whose origins are in the late Cretaceous), a water-circulating system, sterile air, and a specially designed Carbotrap through which all air was passed as exhaust and volatile were hydrocarbons trapped. The fungus for initial testing was Annulohypoxylon sp., isolated as an endophyte of Citrus aurantifolia. It produces, in solid and liquid media, a series of hydrocarbon-like molecules. Some of these including 1,8-cineole, 2-butanone, propanoic acid, 2-methyl-, methyl ester, benzene (1-methylethyl)-, phenylethyl alcohol, benzophenone and azulene, 1,2,3,5,6,7,8,8a-octahydro-1,4-dimethyl-7-(1-methylethenyl), [1S-(1α,7α,8aβ)]. These were the key signature compounds used in an initial Paleobiosphere test. After 3 weeks, incubation, the volatiles associated with the harvested "Trap Shale" included each of the signature substances as well as other fungal-associated products: some indanes, benzene derivatives, some cyclohexanes, 3-octanone, naphthalenes and others. The fungus thus produced a series of "Trap Shale" products that were representative of each of the major classes of hydrocarbons in diesel fuel (Mycodiesel). Initial tests with the Paleobiosphere offer some evidence for a possible origin of hydrocarbons trapped in bentonite shale. Thus, with modifications, numerous other tests can also be designed for utilization in the Paleobiosphere.

  18. Electron-ion recombination in laser-produced plasmas using optical interferometry

    NASA Astrophysics Data System (ADS)

    Heilmann, Nathan; Peatross, Justin; Bergeson, Scott

    2011-10-01

    We are developing methods to measure electron-ion recombination in laser-produced plasmas. A high intensity fs laser pulse is focused into a gas jet and forms a plasma. A weaker probe beam first passes through a slightly mis-aligned Michelson interferometer and is also focused into the plasma. The probe ``beam'' is actually two temporally coincident but spatially offset laser beams. One of the laser beams passes through the plasma and the other does not. These beams expand and produce interference fringes in the far field, similar to a Young's double slit experiment. The spatial position of these fringes depends on the differential phase shift in the two probe beams. This differential shift is due to the electron density in the plasma, which is probed by only one beam. By measuring the fringe shift as a function of time after the plasma is formed, we should be able to measure the time-evolving electron density. At sufficiently high densities, three-body recombination will become important. In that regime, the measured recombination rate can be used to determine the electron temperature.

  19. Physicochemical and biological characteristics of the nanostructured polysaccharide-iron hydrogel produced by microorganism Klebsiella oxytoca.

    PubMed

    Kianpour, Sedigheh; Ebrahiminezhad, Alireza; Mohkam, Milad; Tamaddon, Ali Mohammad; Dehshahri, Ali; Heidari, Reza; Ghasemi, Younes

    2017-02-01

    There is an increasing interest in the nanostructured polysaccharide-iron hydrogel produced by Klebsiella oxytoca. Critical physicochemical and biological characteristics of these nanostructures should be revealed for biomedical applications. Accordingly, an iron reducing strain K. oxytoca, which synthesizes biogenic polysaccharide-iron hydrogel nanoparticles, known as Fe (III)-exopolysaccharide (Fe-EPS) was isolated from a mineral spring. For microbiological identification purpose 16S rRNA sequence analysis and different morphological, physiological, and biochemical characteristics of the isolate were studied. Critical physicochemical and biological characteristics of the produced Fe-EPS were evaluated using transmission electron microscopy (TEM), Fourier transform infrared (FTIR) spectroscopy, X-ray crystallography (XRD), vibrating sample magnetometer (VSM). In addition, for the first time, Fe-EPS which synthesized by K. oxytoca was evaluated by dynamic light scattering (DLS), thermo gravimetric analysis (TGA), and cytotoxicity assay. TEM micrographs showed that the biogenic Fe-EPS is composed of ultra-small (about 1.8 nm) iron oxide nanoparticles (IONs) which are trapped in a polysaccharide matrix. The matrix was about 17% (w/w) of Fe-EPS total weight and provided a large negative charge of -71 mV. Interestingly, Fe-EPS showed a growth promotion effect on hepatocarcinoma cell line (Hep-G2) and 36% increase in the percentage of viability was observed by 24 h exposure to 500 μg ml(-1) Fe-EPS.

  20. Preliminary characterization of biosurfactants produced by microorganisms isolated from refinery wastewaters.

    PubMed

    Yalçin, Emine; Ergene, Aysun

    2010-02-01

    Some bacterial strains isolated from refinery wastewaters were identified as Pseudomonas aeruginosa RWI, Pseudomonas putida RWII, Pseudomonas fluorescens RWIII and Burkholderia cepacia RWIV, and the biosurfactants produced by these strains were coded as BS-I, BS-II, BS-III and BS-IV, respectively. The bacterial strains were characterized by the following biochemical methods: Gram stain, oxidase activity, indol, lactose and growth at 42 degrees C. Biosurfactant production was evaluated by: emulsification activity, surface tension measurement and critical micelle concentration. Chemical characterization of the biosurfactants was done by: FTIR and analysis of carbohydrate, protein and lipid content. The biosurfactants showed good emulsification activity against different hydrocarbon sources. The initial surface tension of culture broth was determined as 67.3 mN/m, and production of BS-I, BS-II, BS-III and BS-IV lowered this value to 35.9, 49.2, 51.6 and 45.7 mN/m, respectively. The critical micelle concentration of the biosurfactants was found to be in the range 10-50 mg/L. From the results of this study it was observed that the refinery wastewaters are a suitable source for isolation of biosurfactant-producing bacteria, but are not a substrate for biosurfactant production.

  1. Fluorescence technique for on-line monitoring of state of hydrogen-producing microorganisms

    DOEpatents

    Seibert, Michael; Makarova, Valeriya; Tsygankov, Anatoly A.; Rubin, Andrew B.

    2007-06-12

    In situ fluorescence method to monitor state of sulfur-deprived algal culture's ability to produce H.sub.2 under sulfur depletion, comprising: a) providing sulfur-deprived algal culture; b) illuminating culture; c) measuring onset of H.sub.2 percentage in produced gas phase at multiple times to ascertain point immediately after anerobiosis to obtain H.sub.2 data as function of time; and d) determining any abrupt change in three in situ fluorescence parameters; i) increase in F.sub.t (steady-state level of chlorophyll fluorescence in light adapted cells); ii) decrease in F.sub.m', (maximal saturating light induced fluorescence level in light adapted cells); and iii) decrease in .DELTA.F/F.sub.m'=(F.sub.m'-F.sub.t)/F.sub.m' (calculated photochemical activity of photosystem II (PSII) signaling full reduction of plastoquinone pool between PSII and PSI, which indicates start of anaerobic conditions that induces synthesis of hydrogenase enzyme for subsequent H.sub.2 production that signal oxidation of plastoquinone pool asmain factor to regulate H.sub.2 under sulfur depletion.

  2. Cyt toxins produced by Bacillus thuringiensis: a protein fold conserved in several pathogenic microorganisms.

    PubMed

    Soberón, Mario; López-Díaz, Jazmin A; Bravo, Alejandra

    2013-03-01

    Bacillus thuringiensis bacteria produce different insecticidal proteins known as Cry and Cyt toxins. Among them the Cyt toxins represent a special and interesting group of proteins. Cyt toxins are able to affect insect midgut cells but also are able to increase the insecticidal damage of certain Cry toxins. Furthermore, the Cyt toxins are able to overcome resistance to Cry toxins in mosquitoes. There is an increasing potential for the use of Cyt toxins in insect control. However, we still need to learn more about its mechanism of action in order to define it at the molecular level. In this review we summarize important aspects of Cyt toxins produced by Bacillus thuringiensis, including current knowledge of their mechanism of action against mosquitoes and also we will present a primary sequence and structural comparison with related proteins found in other pathogenic bacteria and fungus that may indicate that Cyt toxins have been selected by several pathogenic organisms to exert their virulence phenotypes. Copyright © 2012 Elsevier Inc. All rights reserved.

  3. Key determinants affecting sheep wool biodegradation directed by a keratinase-producing Bacillus subtilis recombinant strain.

    PubMed

    Zaghloul, Taha I; Embaby, Amira M; Elmahdy, Ahmed R

    2011-02-01

    OVAT (one variable at a time) approach was applied in this study to screen the most important physicochemical key determinants involved in the process of sheep wool biodegradation. The process was directed by a keratinase-producing Bacillus subtilis DB 100 (p5.2) recombinant strain. Data indicate that, sheep wool could be degraded efficiently in cultures incubated at 30°C, with initial pH of 7 with agitation at 150 rpm. Two times autoclaved alkali treated and undefatted chopped sheep wool is more accessible to biodegradation. B. subtilis recombinant cells could utilize sheep wool as a sole source of carbon and nitrogen. Sheep wool-based modified basal medium II, lacking NH₄Cl and yeast extract, could greatly support the growth of these bacterial cells. Sheep wool biodegradation was conducted efficiently in the absence of kanamycin consequently; high stability of the recombinant plasmid (p5.2) represents a great challenge upon scaling up this process. Three key determinants (sheep wool concentration, incubation time and inoculum size) imposing considerable constraints on the process are highlighted. Sheep wool-based tap water medium and sheep wool-based distilled water medium were formulated in this study. High levels of released end products, produced from sheep wool biodegradation are achieved upon using these two sheep wool-based water media. Data indicate that, sheep wool hydrolysate is rich in some amino acids, such as tyrosine, phenylalanine, lysine, proline, isoleucine, leucine, valine, aspartic acid and glutamic acid. Moreover, the resulting sheep wool hydrolysate contains soluble proteins of high and intermediate molecular weights. The present study demonstrates a feasible, cheap, reproducible, efficient and rapid biotechnological approach towards utilization of raw sheep wool waste through a recombinant bacterium.

  4. Generation of a Chinese Hamster Ovary Cell Line Producing Recombinant Human Glucocerebrosidase

    PubMed Central

    Novo, Juliana Branco; Morganti, Ligia; Moro, Ana Maria; Paes Leme, Adriana Franco; Serrano, Solange Maria de Toledo; Raw, Isaias; Ho, Paulo Lee

    2012-01-01

    Impaired activity of the lysosomal enzyme glucocerebrosidase (GCR) results in the inherited metabolic disorder known as Gaucher disease. Current treatment consists of enzyme replacement therapy by administration of exogenous GCR. Although effective, it is exceptionally expensive, and patients worldwide have a limited access to this medicine. In Brazil, the public healthcare system provides the drug free of charge for all Gaucher's patients, which reaches the order of $ 84 million per year. However, the production of GCR by public institutions in Brazil would reduce significantly the therapy costs. Here, we describe a robust protocol for the generation of a cell line producing recombinant human GCR. The protein was expressed in CHO-DXB11 (dhfr−) cells after stable transfection and gene amplification with methotrexate. As expected, glycosylated GCR was detected by immunoblotting assay both as cell-associated (~64 and 59 kDa) and secreted (63–69 kDa) form. Analysis of subclones allowed the selection of stable CHO cells producing a secreted functional enzyme, with a calculated productivity of 5.14 pg/cell/day for the highest producer. Although being laborious, traditional methods of screening high-producing recombinant cells may represent a valuable alternative to generate expensive biopharmaceuticals in countries with limited resources. PMID:23091360

  5. Response of soil microorganisms to As-produced and functionalized single-wall carbon nanotubes (SWNTs).

    PubMed

    Tong, Zhonghua; Bischoff, Marianne; Nies, Loring F; Myer, Phillip; Applegate, Bruce; Turco, Ronald F

    2012-12-18

    The use of single-wall carbon nanotubes (SWNTs) in manufacturing and biomedical applications is increasing at a rapid rate; however data on the effects of a potential environmental release of the materials remain sparse. In this study, soils with either low or high organic matter contents as well as pure cultures of E. coli are challenged with either raw as-produced SWNTs (AP-SWNTs) or SWNTs functionalized with either polyethyleneglycol (PEG-SWNTs) or m-polyaminobenzene sulfonic acid (PABS-SWNTs). To mimic chronic exposure, the soil systems were challenged weekly for six weeks; microbial activities and community structures for both the prokaryote and eukaryote community were evaluated. Results show that repeated applications of AP-SWNTs can affect microbial community structures and induce minor changes in soil metabolic activity in the low organic matter systems. Toxicity of the three types of SWNTs was also assessed in liquid cultures using a bioluminescent E. coli-O157:H7 strain. Although decreases in light were detected in all treated samples, low light recovery following glucose addition in AP-SWNTs treatment and light absorption property of SWNTs particles suggest that AP-SWNTs suppressed metabolic activity of the E. coli, whereas the two functionalized SWNTs are less toxic. The metals released from the raw forms of SWNTs would not play a role in the effects seen in soil or the pure culture. We suggest that sorption to soil organic matter plays a controlling role in the soil microbiological responses to these nanomaterials.

  6. Genome Sequence of the Streptomycin-Producing Microorganism Streptomyces griseus IFO 13350▿ †

    PubMed Central

    Ohnishi, Yasuo; Ishikawa, Jun; Hara, Hirofumi; Suzuki, Hirokazu; Ikenoya, Miwa; Ikeda, Haruo; Yamashita, Atsushi; Hattori, Masahira; Horinouchi, Sueharu

    2008-01-01

    We determined the complete genome sequence of Streptomyces griseus IFO 13350, a soil bacterium producing an antituberculosis agent, streptomycin, which is the first aminoglycoside antibiotic, discovered more than 60 years ago. The linear chromosome consists of 8,545,929 base pairs (bp), with an average G+C content of 72.2%, predicting 7,138 open reading frames, six rRNA operons (16S-23S-5S), and 66 tRNA genes. It contains extremely long terminal inverted repeats (TIRs) of 132,910 bp each. The telomere's nucleotide sequence and secondary structure, consisting of several palindromes with a loop sequence of 5′-GGA-3′, are different from those of typical telomeres conserved among other Streptomyces species. In accordance with the difference, the chromosome has pseudogenes for a conserved terminal protein (Tpg) and a telomere-associated protein (Tap), and a novel pair of Tpg and Tap proteins is instead encoded by the TIRs. Comparisons with the genomes of two related species, Streptomyces coelicolor A3(2) and Streptomyces avermitilis, clarified not only the characteristics of the S. griseus genome but also the existence of 24 Streptomyces-specific proteins. The S. griseus genome contains 34 gene clusters or genes for the biosynthesis of known or unknown secondary metabolites. Transcriptome analysis using a DNA microarray showed that at least four of these clusters, in addition to the streptomycin biosynthesis gene cluster, were activated directly or indirectly by AdpA, which is a central transcriptional activator for secondary metabolism and morphogenesis in the A-factor (a γ-butyrolactone signaling molecule) regulatory cascade in S. griseus. PMID:18375553

  7. The Functional Quality of Soluble Recombinant Polypeptides Produced in Escherichia coli Is Defined by a Wide Conformational Spectrum▿

    PubMed Central

    Martínez-Alonso, Mónica; González-Montalbán, Nuria; García-Fruitós, Elena; Villaverde, Antonio

    2008-01-01

    We have observed that a soluble recombinant green fluorescent protein produced in Escherichia coli occurs in a wide conformational spectrum. This results in differently fluorescent protein fractions in which morphologically diverse soluble aggregates abound. Therefore, the functional quality of soluble versions of aggregation-prone recombinant proteins is defined statistically rather than by the prevalence of a canonical native structure. PMID:18836021

  8. Recombinant L-Asparaginase from Zymomonas mobilis: A Potential New Antileukemic Agent Produced in Escherichia coli

    PubMed Central

    Pereira, Juliana Christina Castanheira Vicente; Costa-Amaral, Isabele Campos; da Costa, Elaine Sobral; Ribeiro, Maria Cecília Menks; Land, Marcelo Gerardin Poirot; Alves, Tito Lívio Moitinho; Larentis, Ariane Leites; Almeida, Rodrigo Volcan

    2016-01-01

    L-asparaginase is an enzyme used as a chemotherapeutic agent, mainly for treating acute lymphoblastic leukemia. In this study, the gene of L-asparaginase from Zymomonas mobilis was cloned in pET vectors, fused to a histidine tag, and had its codons optimized. The L-asparaginase was expressed extracellularly and intracellularly (cytoplasmically) in Escherichia coli in far larger quantities than obtained from the microorganism of origin, and sufficient for initial cytotoxicity tests on leukemic cells. The in silico analysis of the protein from Z. mobilis indicated the presence of a signal peptide in the sequence, as well as high identity to other sequences of L-asparaginases with antileukemic activity. The protein was expressed in a bioreactor with a complex culture medium, yielding 0.13 IU/mL extracellular L-asparaginase and 3.6 IU/mL intracellular L-asparaginase after 4 h of induction with IPTG. The cytotoxicity results suggest that recombinant L-asparaginase from Z. mobilis expressed extracellularly in E.coli has a cytotoxic and cytostatic effect on leukemic cells. PMID:27253887

  9. Recombinant L-Asparaginase from Zymomonas mobilis: A Potential New Antileukemic Agent Produced in Escherichia coli.

    PubMed

    Einsfeldt, Karen; Baptista, Isis Cavalcante; Pereira, Juliana Christina Castanheira Vicente; Costa-Amaral, Isabele Campos; Costa, Elaine Sobral da; Ribeiro, Maria Cecília Menks; Land, Marcelo Gerardin Poirot; Alves, Tito Lívio Moitinho; Larentis, Ariane Leites; Almeida, Rodrigo Volcan

    2016-01-01

    L-asparaginase is an enzyme used as a chemotherapeutic agent, mainly for treating acute lymphoblastic leukemia. In this study, the gene of L-asparaginase from Zymomonas mobilis was cloned in pET vectors, fused to a histidine tag, and had its codons optimized. The L-asparaginase was expressed extracellularly and intracellularly (cytoplasmically) in Escherichia coli in far larger quantities than obtained from the microorganism of origin, and sufficient for initial cytotoxicity tests on leukemic cells. The in silico analysis of the protein from Z. mobilis indicated the presence of a signal peptide in the sequence, as well as high identity to other sequences of L-asparaginases with antileukemic activity. The protein was expressed in a bioreactor with a complex culture medium, yielding 0.13 IU/mL extracellular L-asparaginase and 3.6 IU/mL intracellular L-asparaginase after 4 h of induction with IPTG. The cytotoxicity results suggest that recombinant L-asparaginase from Z. mobilis expressed extracellularly in E.coli has a cytotoxic and cytostatic effect on leukemic cells.

  10. The potential of transgenic green microalgae; a robust photobioreactor to produce recombinant therapeutic proteins.

    PubMed

    Akbari, Fariba; Eskandani, Morteza; Khosroushahi, Ahmad Yari

    2014-11-01

    Microalgae have been used in food, cosmetic, and biofuel industries as a natural source of lipids, vitamins, pigments and antioxidants for a long time. Green microalgae, as potent photobioreactors, can be considered as an economical expression system to produce recombinant therapeutical proteins at large-scale due to low cost of production and scaling-up capitalization owning to the inexpensive medium requirement, fast growth rate, and the ease of manipulation. These microalgae possess all benefit eukaryotic expression systems including the ability of post-translational modifications required for proper folding and stability of active proteins. Among the many items regarded as recombinant protein production, this review compares the different expression systems with green microalgae like Dunaliella by viewing the nuclear/chloroplast transformation challenges/benefits, related selection markers/reporter genes, and crucial factors/strategies affecting the increase of foreign protein expression in microalgae transformants. Some important factors were discussed regarding the increase of protein yielding in microalgae transformants including: transformation-associated genotypic modifications, endogenous regulatory factors, promoters, codon optimization, enhancer elements, and milking of recombinant protein.

  11. Biological treatment of produced water in a sequencing batch reactor by a consortium of isolated halophilic microorganisms.

    PubMed

    Pendashteh, A R; Fakhru'l-Razi, A; Chuah, T G; Radiah, A B Dayang; Madaeni, S S; Zurina, Z A

    2010-10-01

    Produced water or oilfield wastewater is the largest volume ofa waste stream associated with oil and gas production. The aim of this study was to investigate the biological pretreatment of synthetic and real produced water in a sequencing batch reactor (SBR) to remove hydrocarbon compounds. The SBR was inoculated with isolated tropical halophilic microorganisms capable of degrading crude oil. A total sequence of 24 h (60 min filling phase; 21 h aeration; 60 min settling and 60 min decant phase) was employed and studied. Synthetic produced water was treated with various organic loading rates (OLR) (0.9 kg COD m(-3) d(-1), 1.8 kg COD m(-3) d(-1) and 3.6 kg COD m(-3) d(-1)) and different total dissolved solids (TDS) concentration (35,000 mg L(-1), 100,000 mg L(-1), 150,000 mg L(-1), 200,000 mg L(-1) and 250,000 mg L(-1)). It was found that with an OLR of 0.9 kg COD m(-3) d(-1) and 1.8 kg COD m(-3) d(-1), average oil and grease (O&G) concentrations in the effluent were 7 mg L(-1) and 12 mg L(-1), respectively. At TDS concentration of 35,000 mg L(-1) and at an OLR of 1.8 kg COD m(-3)d(-1), COD and O&G removal efficiencies were more than 90%. However, with increase in salt content to 250,000 mg L(-1), COD and O&G removal efficiencies decreased to 74% and 63%, respectively. The results of biological treatment of real produced water showed that the removal rates of the main pollutants of wastewater, such as COD, TOC and O&G, were above 81%, 83%, and 85%, respectively.

  12. Study of recombinant micro-organism populations characterized by their plasmid content per cell using a segregated model.

    PubMed

    Shene, C; Andrews, B A; Asenjo, J A

    2003-07-01

    Numerous observations from recombinant systems have shown that properties such as the specific cell growth rate and the plasmid-free cell formation rate are related, not only to the average plasmid content per cell, but also to the plasmid distribution within a population. The plasmid distribution in recombinant cultures can have an effect on the culture productivity that cannot be modelled using average values of the overall culture. The prediction of the behaviour of a plasmid content distribution and its causes and effects can only be studied using segregated models. A segregated model that describes populations of recombinant cells characterized by their plasmid content distribution has been developed. This model includes critical causes of recombinant culture instability such as the plasmid partition mechanism at cell division, plasmid replication kinetics and the effect of the plasmid content on the specific growth rate. The segregated model allows investigation of the effect of each of these causes and that of the plasmid content distribution on the observable behaviour of a recombinant culture. The effect of two partitioning mechanisms (Gaussian distribution and binomial distribution) on culture stability was investigated. The Gaussian distribution is slightly more stable. A small plasmid replication rate constant results in a very unstable culture even after short periods of time. This instability is dramatically improved for a larger value of this constant, hence improving protein synthesis. For a very narrow initial plasmid distribution, a given plasmid replication rate and partitioning mechanism can become broad even after a relatively short period of time. In contrast, a very "broad" initial distribution gave rise to a "Gamma-like" distribution profile. If we compare the results obtained in the simulations of the segregated model with those of the non-segregated one (average model), the latter model predicts much more stable behaviour, thus these

  13. Efficient preservation in a silicon oxide matrix of Escherichia coli, producer of recombinant proteins.

    PubMed

    Desimone, Martín F; De Marzi, Mauricio C; Copello, Guillermo J; Fernández, Marisa M; Malchiodi, Emilio L; Diaz, Luis E

    2005-10-01

    The aim of this work was to study the use of silicon oxide matrices for the immobilization and preservation of recombinant-protein-producing bacteria. We immobilized Escherichia coli BL21 transformants containing different expression plasmids. One contained DNA coding for a T-cell receptor beta chain, which was expressed as inclusion bodies in the cytoplasm. The other two encoded bacterial superantigens Staphylococcal Enterotoxin G and Streptococcal Superantigen, which were expressed as soluble proteins in the periplasm. The properties of immobilization and storage stability in inorganic matrices prepared from two precursors, silicon dioxide and tetraethoxysilane, were studied. Immobilized E. coli was stored in sealed tubes at 4 and 20 degrees C and the number of viable cells and level of recombinant protein production were analyzed weekly. Different tests showed that the biochemical characteristics of immobilized E. coli remained intact. At both temperatures selected, we found that the number of bacteria in silicon dioxide-derived matrix was of the same order of magnitude (10(9) cfu ml(-1)) as before immobilization, for 2 months. After 2 weeks, cells immobilized in an alkoxide-derived matrix decreased to 10(4) cfu ml(-1) at 4 degrees C, and no viable cells were detected at 20 degrees C. We found that immobilized bacteria could be used as a starter to produce recombinant proteins with yields comparable to those obtained from glycerol stocks: 15 mg l(-1) for superantigens and 2 mg l(-1) for T-cell receptor beta chain. These results contribute to the development of methods for microbial cell preservation under field conditions.

  14. Synthesis of nylon 4 from gamma-aminobutyrate (GABA) produced by recombinant Escherichia coli.

    PubMed

    Park, Si Jae; Kim, Eun Young; Noh, Won; Oh, Young Hoon; Kim, Hye Young; Song, Bong Keun; Cho, Kwang Myung; Hong, Soon Ho; Lee, Seung Hwan; Jegal, Jonggeon

    2013-07-01

    In this study, we developed recombinant Escherichia coli strains expressing Lactococcus lactis subsp. lactis Il1403 glutamate decarboxylase (GadB) for the production of GABA from glutamate monosodium salt (MSG). Syntheses of GABA from MSG were examined by employing recombinant E. coli XL1-Blue as a whole cell biocatalyst in buffer solution. By increasing the concentration of E. coli XL1-Blue expressing GadB from the OD₆₀₀ of 2-10, the concentration and conversion yield of GABA produced from 10 g/L of MSG could be increased from 4.3 to 4.8 g/L and from 70 to 78 %, respectively. Furthermore, E. coli XL1-Blue expressing GadB highly concentrated to the OD₆₀₀ of 100 produced 76.2 g/L of GABA from 200 g/L of MSG with 62.4 % of GABA yield. Finally, nylon 4 could be synthesized by the bulk polymerization using 2-pyrrolidone that was prepared from microbially synthesized GABA by the reaction with Al₂O₃ as catalyst in toluene with the yield of 96 %.

  15. Recombination of engineered defective RNA species produces infective potyvirus in planta.

    PubMed

    Gal-On, A; Meiri, E; Raccah, B; Gaba, V

    1998-06-01

    Recombination occurred between viral genomes when squash plants were cobombarded with mixtures of engineered disabled constructs of a zucchini yellow mosaic potyvirus. Single and double recombinants were detected in the progeny. Genes involved in the recombination process and the mechanisms of recombination were studied in potyviruses for the first time.

  16. Recombination of Engineered Defective RNA Species Produces Infective Potyvirus In Planta†

    PubMed Central

    Gal-On, Amit; Meiri, Eti; Raccah, Benjamin; Gaba, Victor

    1998-01-01

    Recombination occurred between viral genomes when squash plants were cobombarded with mixtures of engineered disabled constructs of a zucchini yellow mosaic potyvirus. Single and double recombinants were detected in the progeny. Genes involved in the recombination process and the mechanisms of recombination were studied in potyviruses for the first time. PMID:9573302

  17. Constructing a recombinant hyaluronic acid biosynthesis operon and producing food-grade hyaluronic acid in Lactococcus lactis.

    PubMed

    Sheng, Juzheng; Ling, Peixue; Wang, Fengshan

    2015-02-01

    Hyaluronic acid (HA), a natural high molecular weight polysaccharide, is produced by Streptococcus zooepidemicus. However, Streptococcus has several drawbacks including its potential to produce exotoxins, so there is demand for an alternative HA source. Here, a recombinant HA biosynthesis operon, as well as the HA biosynthesis operon of S. zooepidemicus were introduced into L. lactis using the nisin-controlled expression system, respectively. HA was successfully synthesized by recombinant L. lactis. Furthermore, overexpression of the endogenous enzymes directing the synthesis of precursor sugars was effective at increasing HA production, and increasing the supply of UDP-activated monosaccharide donors aided synthesis of monodisperse HA polysaccharides. Besides GRAS host strain (L. lactis) and NICE system, the selecting marker (lacF gene) of the recombinant strain is also food grade. Therefore, HA produced by recombinant L. lactis overcomes the problems associated with Streptococcus and provides a source of food-grading HA appropriate for widespread biotechnological applications.

  18. Specific CEA-producing colorectal carcinoma cell killing with recombinant adenoviral vector containing cytosine deaminase gene

    PubMed Central

    Shen, Li-Zong; Wu, Wen-Xi; Xu, De-Hua; Zheng, Zhong-Cheng; Liu, Xin-Yuan; Ding, Qiang; Hua, Yi-Bing; Yao, Kun

    2002-01-01

    AIM: To kill CEA positive colorectal carcinoma cells specifically using the E coli cytosine deaminase (CD) suicide gene, a new replication-deficient recombinant adenoviral vector was constructed in which CD gene was controlled under CEA promoter and its in vitro cytotoxic effects were evaluated. METHODS: Shuttle plasmid containing CD gene and regulatory sequence of the CEA gene was constructed and recombined with the right arm of adenovirus genome DNA in 293 cell strain. Dot blotting and PCR were used to identify positive plaques. The purification of adenovirus was performed with ultra-concentration in CsCl step gradients and the titration was measured with plaque formation assay. Cytotoxic effects were assayed with MTT method, The fifty percent inhibition concentration (IC50) of 5-FC was calculated using a curve-fitting parameter. The human colorectal carcinoma cell line, which was CEA-producing, and the CEA-nonproducing Hela cell line were applied in cytological tests. An established recombinant adenovirus vector AdCMVCD, in which the CD gene was controlled under CMV promoter, was used as virus control. Quantitative results were expressed as the mean ± SD of the mean. Statistical analysis was performed using ANOVA test. RESULTS: The desired recombinant adenovirus vector was named AdCEACD. The results of dot blotting and PCR showed that the recombinant adenovirus contained CEA promoter and CD gene. Virus titer was about 5.0 × 1014 pfu/L-1 after purification. The CEA-producing Lovo cells were sensitive to 5-FC and had the same cytotoxic effect after infection with AdCEACD and AdCMVCD (The IC50 values of 5-FC in parent Lovo cells, Lovo cells infected with 100 M.O.I AdCEACD and Lovo cells infected with 10 M.O.I AdCMVCD were > 15000, 216.5 ± 38.1 and 128.8 ± 25.4 μmol•L⁻¹, P < 0.001, respectively), and the cytotoxicity of 5-FC increased accordingly when the M.O.I of adenoviruses were enhanced (The value of IC50 of 5-FC was reduced to 27.9 ± 4.2 μmol•L-1

  19. QSAR study and the hydrolysis activity prediction of three alkaline lipases from different lipase-producing microorganisms.

    PubMed

    Wang, Haikuan; Wang, Xiaojie; Li, Xiaolu; Zhang, Yehong; Dai, Yujie; Guo, Changlu; Zheng, Heng

    2012-09-28

    The hydrolysis activities of three alkaline lipases, L-A1, L-A2 and L-A3 secreted by different lipase-producing microorganisms isolated from the Bay of Bohai, P. R. China were characterized with 16 kinds of esters. It was found that all the lipases have the ability to catalyze the hydrolysis of the glycerides, methyl esters, ethyl esters, especially for triglycerides, which shows that they have broad substrate spectra, and this property is very important for them to be used in detergent industry. Three QSAR models were built for L-A1, L-A2 and L-A3 respectively with GFA using Discovery studio 2.1. The models equations 1, 2 and 3 can explain 95.80%, 97.45% and 97.09% of the variances (R(2)(adj)) respectively while they could predict 95.44%, 89.61% and 93.41% of the variances (R(2)(cv)) respectively. With these models the hydrolysis activities of these lipases to mixed esters were predicted and the result showed that the predicted values are in good agreement with the measured values, which indicates that this method can be used as a simple tool to predict the lipase activities for single or mixed esters.

  20. QSAR study and the hydrolysis activity prediction of three alkaline lipases from different lipase-producing microorganisms

    PubMed Central

    2012-01-01

    The hydrolysis activities of three alkaline lipases, L-A1, L-A2 and L-A3 secreted by different lipase-producing microorganisms isolated from the Bay of Bohai, P. R. China were characterized with 16 kinds of esters. It was found that all the lipases have the ability to catalyze the hydrolysis of the glycerides, methyl esters, ethyl esters, especially for triglycerides, which shows that they have broad substrate spectra, and this property is very important for them to be used in detergent industry. Three QSAR models were built for L-A1, L-A2 and L-A3 respectively with GFA using Discovery studio 2.1. The models equations 1, 2 and 3 can explain 95.80%, 97.45% and 97.09% of the variances (R2adj) respectively while they could predict 95.44%, 89.61% and 93.41% of the variances (R2cv) respectively. With these models the hydrolysis activities of these lipases to mixed esters were predicted and the result showed that the predicted values are in good agreement with the measured values, which indicates that this method can be used as a simple tool to predict the lipase activities for single or mixed esters. PMID:23016923

  1. Inhibition of microorganisms involved in deterioration of an archaeological site by silver nanoparticles produced by a green synthesis method.

    PubMed

    Carrillo-González, Rogelio; Martínez-Gómez, Miriam Araceli; González-Chávez, Ma Del Carmen A; Mendoza Hernández, José Carlos

    2016-09-15

    The Citadel, part of the pre-Hispanic city of Teotihuacan and listed as a World Heritage Site, harbors irreplaceable archaeological walls and murals. This city was abandoned by the 7th century and its potential deterioration represents a noteworthy loss of the world's cultural heritage. This research consisted of isolation and identification of bacteria and fungi contributing to this deterioration from walls of a pre-Hispanic city. In addition, silver nanoparticles (AgNP) produced, using a green synthesis method, were tested as potential inhibitors of microbes. AgNP of different sizes and concentrations were tested using in situ assays. Leaf aqueous extracts from two plants species (Foeniculum vulgare and Tecoma stans) and two extraction procedures were used in the NP synthesis. The potential of AgNP as preventive/corrective treatments to protect stucco materials from biodeterioration, as well as the microbial inhibition on three stone materials (stucco, basalt and calcite) was analyzed. Twenty-three bacterial species belonging to eight genera and fourteen fungal species belonging to seven genera were isolated from colored stains, patinas and biofilms produced on the surfaces of archaeological walls from the pre-Hispanic city, Teotihuacan. AgNP from F. vulgare were more effective for in vitro microbial growth inhibition than those from T. stans. Bacteria were less sensitive to AgNP than fungi; however, sensitivity mainly depended on the microbial strain and the plant extract used to prepare AgNP. The use of AgNP as a preventive or corrective treatment to decrease microbial colonization in three kinds of stone used in historical walls was successful. Calcite was more colonized by Alternaria alternata, but less by Pectobacterium carotovorum. This is the first study at different scales (in vitro and tests on different stone types) of inhibition of biodeterioration-causing microorganisms isolated from an archaeological site by green synthesized AgNP.

  2. Purification and basic biochemical characterization of 19 recombinant plant peroxidase isoenzymes produced in Pichia pastoris☆

    PubMed Central

    Krainer, Florian W.; Pletzenauer, Robert; Rossetti, Laura; Herwig, Christoph; Glieder, Anton; Spadiut, Oliver

    2014-01-01

    The plant enzyme horseradish peroxidase (HRP) is used in several important industrial and medical applications, of which especially biosensors and diagnostic kits describe an emerging field. Although there is an increasing demand for high amounts of pure enzyme preparations, HRP is still isolated from the plant as a mixture of different isoenzymes with different biochemical properties. Based on a recent next generation sequencing approach of the horseradish transcriptome, we produced 19 individual HRP isoenzymes recombinantly in the yeast Pichia pastoris. After optimizing a previously reported 2-step purification strategy for the recombinant isoenzyme HRP C1A by substituting an unfavorable size exclusion chromatography step with an anion exchange step using a monolithic column, we purified the 19 HRP isoenzymes with varying success. Subsequent basic biochemical characterization revealed differences in catalytic activity, substrate specificity and thermal stability of the purified HRP preparations. The preparations of the isoenzymes HRP A2A and HRP A2B were found to be highly interesting candidates for future applications in diagnostic kits with increased sensitivity. PMID:24342173

  3. [A study of recombinant human sestrin 1 and sestrin 2 proteins produced in a prokaryotic system].

    PubMed

    Rai, N; Kumar, R; Haque, Md A; Hassan, Md I; Dey, S

    2017-01-01

    Sestrins are highly conserved stress-inducible proteins capable of suppressing the production of ROS and signalling through mTORC1. Here we report a study of human sestrin1 (sesn1) and sestrin2 (sesn2) proteins produced in a pET28^(+) vector based prokaryotic system. Mass spectrometry analysis, western blot and surface plasmon resonance (SPR) of affinity purified sesn1 and sesn2 proteins confirmed their identity; biophysical characteristics were observed using circular dichroism (CD) showing that sesn1 and sesn2 have a predominant α-helical structure. Here we describe a simple, one step purification process to purify a large amount of sestrin proteins with significant yield. Further study of recombinant human sestrins may further facilitate the understanding of their roles in eukaryotic cells.

  4. Biological Activity of Recombinant Bovine Interferon τ Produced by a Silkworm-Baculovirus Gene Expression System

    PubMed Central

    TAKAHASHI, Hitomi; TSUNAZAKI, Makoto; HAMANO, Takashi; TAKAHASHI, Masashi; OKUDA, Kiyoshi; INUMARU, Shigeki; OKANO, Akira; GESHI, Masaya; HIRAKO, Makoto

    2013-01-01

    ABSTRACT Bovine interferon (bIFN) τ plays a crucial role in maternal-fetal recognition and was expressed using a Bombyx mori (Bm) nuclear polyhedrosis virus (silkworm baculovirus) gene expression system. The biological effects of Bm-recombinant bIFNτ (rbIFNτ) on prostaglandin (PG) F2α synthesis were investigated in cultured bovine endometrial epithelial cells with oxytocin (OT, 100 nM) and on the in vitro development of bovine embryos. Bm-rbIFNτ and OT were shown to suppress PGF2α production in a dose-dependent manner. When in vitro produced morula stage embryos were cultured for 72 hr in modified CR1aa medium supplemented with or without rbIFNτ, Bm-rbIFNτ (10 ng/ml) significantly promoted development to the expanded blastocyst stage. In conclusion, Bm-rbIFNτ was suggested to have the same bioactivity as native IFNτ. PMID:24212505

  5. Recombinant human milk fat globule-EGF factor 8 produces dose-dependent benefits in sepsis.

    PubMed

    Shah, Kavin G; Wu, Rongqian; Jacob, Asha; Molmenti, Ernesto P; Nicastro, Jeffrey; Coppa, Gene F; Wang, Ping

    2012-01-01

    Animal milk fat globule-EGF factor 8 (MFG-E8) has been shown to be beneficial in attenuating the inflammatory response in sepsis. In this study, we examined the effect of recombinant human MFG-E8 (rhMFG-E8) in an animal model of sepsis in an effort to develop it as a potential therapy against sepsis in humans. Rats were subjected to sepsis by cecal ligation and puncture (CLP), and at 5 h post-CLP, they were given different doses of rhMFG-E8 (20, 40, 80, 160 μg/kg BW) in normal saline. At 20 h post-CLP, samples were collected for further analysis. A 10-day survival study was also performed. At 20 h after CLP, organ injury indicators, serum IL-6 and TNF-α, and plasma HMGB-1 levels were significantly increased as compared to sham-operated animals. Treatment with 20 μg/kg rhMFG-E8 significantly reduced these levels. With higher doses, further reductions in AST and ALT (59-62%), creatinine (65-68%), and lactate (46-57%), and serum IL-6 and TNF-α were obtained. The 160 μg/kg dose produced the greatest reduction in serum TNF-α. With treatment with 20 μg/kg rhMFG-E8, HMGB-1 levels decreased by 80%, returning back to sham values. In a 10-day survival study, vehicle-treated animals produced a 36% survival rate, while rhMFG-E8 significantly improved the survival rate to 68-72%. Treatment with increasing doses of rhMFG-E8 significantly reduced the number of apoptotic cells detected and markedly attenuated the tissue damages observed in the lungs. These data suggest that recombinant human MFG-E8 is beneficial in ameliorating sepsis in an animal model of sepsis.

  6. Diversity of cultivable β-glycosidase-producing micro-organisms isolated from the soil of a ginseng field and their ginsenosides-hydrolysing activity.

    PubMed

    Fu, Y; Yin, Z; Wu, L; Yin, C

    2014-02-01

    This research aimed to explore the diversity of cultivable β-glycosidase-producing micro-organisms in ginseng field soil. Fifty-three strains showing β-glucosidase activity were isolated from a ginseng field, using a newly designed Esculin-R2A agar. All the isolated strains belonged to the genus Agrobacterium, Arthrobacter, Burkholderia, Dyella, Edaphobacter, Luteibacter, Mucilaginibacter, Paenibacillus, Phenylobacterium, Pseudomonas, Sphingomonas and Streptomyces. The main β-glucosidase-producing micro-organisms in the ginseng field soil were Sphingomonas, Burkholderia, Luteibacter and Streptomyces, while concentrations of Agrobacterium, Arthrobacter, Paenibacillus and Pseudomonas were relatively low. Of these micro-organisms, the strain GS 09 could hydrolyse major ginsenosides Rb1, Rb2 and Rc to the active metabolite compound K. The strain GS 09 belonged to the genus Sphingomonas, and its 16S rRNA gene sequence showed 100% similarities with that of Sphingomonas asaccharolytica. This is the first study to provide information of cultivable β-glycosidase-producing micro-organisms in ginseng field soil. The strain GS 09 has potential to be applied on the preparation for minor ginsenoside C-K in pharmaceutical industry. © 2013 The Society for Applied Microbiology.

  7. Transplantation of genetically engineered cardiac fibroblasts producing recombinant human erythropoietin to repair the infarcted myocardium

    PubMed Central

    Ruvinov, Emil; Sharabani-Yosef, Orna; Nagler, Arnon; Einbinder, Tom; Feinberg, Micha S; Holbova, Radka; Douvdevani, Amos; Leor, Jonathan

    2008-01-01

    Background Erythropoietin possesses cellular protection properties. The aim of the present study was to test the hypothesis that in situ expression of recombinant human erythropoietin (rhEPO) would improve tissue repair in rat after myocardial infarction (MI). Methods and results RhEPO-producing cardiac fibroblasts were generated ex vivo by transduction with retroviral vector. The anti-apoptotic effect of rhEPO-producing fibroblasts was evaluated by co-culture with rat neonatal cardiomyocytes exposed to H2O2-induced oxidative stress. Annexin V/PI assay and DAPI staining showed that compared with control, rhEPO forced expression markedly attenuated apoptosis and improved survival of cultured cardiomyocytes. To test the effect of rhEPO on the infarcted myocardium, Sprague-Dawley rats were subjected to permanent coronary artery occlusion, and rhEPO-producing fibroblasts, non-transduced fibroblasts, or saline, were injected into the scar tissue seven days after infarction. One month later, immunostaining identified rhEPO expression in the implanted engineered cells but not in controls. Compared with non-transduced fibroblasts or saline injection, implanted rhEPO-producing fibroblasts promoted vascularization in the scar, and prevented cell apoptosis. By two-dimensional echocardiography and postmortem morphometry, transplanted EPO-engineered fibroblasts did not prevent left ventricular (LV) dysfunction and adverse LV remodeling 5 and 9 weeks after MI. Conclusion In situ expression of rhEPO enhances vascularization and reduces cell apoptosis in the infarcted myocardium. However, local EPO therapy is insufficient for functional improvement after MI in rat. PMID:19014419

  8. Lifetime and quenching of CO /a super 3 pi/ produced by recombination of CO2 ions in a helium afterglow.

    NASA Technical Reports Server (NTRS)

    Wauchop, T. S.; Broida, H. P.

    1972-01-01

    Demonstration that rapid dissociative recombination of CO2(+) in a flowing, helium afterglow is an efficient source of CO in the a super 3 pi metastable state. Ions produced by mixing CO2 with He(2 super 3 S) recombine to produce a CO metastable afterglow with a number density as great as 10 to the 9th per sq cm. Monitoring of the (a super 3 pi-X super 1 sigma) Cameron transition in CO was used to study the lifetime and quenching of CO (a super 3 pi) by CO2, N2, NO, and He. Recombination of CO2(+) also produces CO in the d super 3 delta and a' super 3 sigma states.

  9. Manufacturing process used to produce long-acting recombinant factor VIII Fc fusion protein.

    PubMed

    McCue, Justin; Kshirsagar, Rashmi; Selvitelli, Keith; Lu, Qi; Zhang, Mingxuan; Mei, Baisong; Peters, Robert; Pierce, Glenn F; Dumont, Jennifer; Raso, Stephen; Reichert, Heidi

    2015-07-01

    Recombinant factor VIII Fc fusion protein (rFVIIIFc) is a long-acting coagulation factor approved for the treatment of hemophilia A. Here, the rFVIIIFc manufacturing process and results of studies evaluating product quality and the capacity of the process to remove potential impurities and viruses are described. This manufacturing process utilized readily transferable and scalable unit operations and employed multi-step purification and viral clearance processing, including a novel affinity chromatography adsorbent and a 15 nm pore size virus removal nanofilter. A cell line derived from human embryonic kidney (HEK) 293H cells was used to produce rFVIIIFc. Validation studies evaluated identity, purity, activity, and safety. Process-related impurity clearance and viral clearance spiking studies demonstrate robust and reproducible removal of impurities and viruses, with total viral clearance >8-15 log10 for four model viruses (xenotropic murine leukemia virus, mice minute virus, reovirus type 3, and suid herpes virus 1). Terminal galactose-α-1,3-galactose and N-glycolylneuraminic acid, two non-human glycans, were undetectable in rFVIIIFc. Biochemical and in vitro biological analyses confirmed the purity, activity, and consistency of rFVIIIFc. In conclusion, this manufacturing process produces a highly pure product free of viruses, impurities, and non-human glycan structures, with scale capabilities to ensure a consistent and adequate supply of rFVIIIFc. Copyright © 2015 Biogen. Published by Elsevier Ltd.. All rights reserved.

  10. Straightforward approach to produce recombinant scorpion toxins-Pore blockers of potassium channels.

    PubMed

    Nekrasova, Oksana; Kudryashova, Ksenia; Fradkov, Arkadiy; Yakimov, Sergey; Savelieva, Maria; Kirpichnikov, Mikhail; Feofanov, Alexey

    2017-01-10

    Scorpion venom peptide blockers (KTx) of potassium channels are a valuable tool for structure-functional studies and prospective candidates for medical applications. Low yields of recombinant KTx hamper their wide application. We developed convenient and efficient bioengineering approach to a large-scale KTx production that meets increasing demands for such peptides. Maltose-binding protein was used as a carrier for cytoplasmic expression of folded disulfide-rich KTx in E. coli. TEV protease was applied for in vitro cleavage of the target peptide from the carrier. To produce KTx with retained native N-terminal sequence, the last residue of TEV protease cleavage site (CSTEV) was occupied by the native N-terminal residue of a target peptide. It was shown that decreased efficiency of hydrolysis of fusion proteins with non-canonical CSTEV can be overcome without by-product formation. Disulfide formation and folding of a target peptide occurred in cytoplasm eliminating the need for renaturation procedure in vitro. Advantages of this approach were demonstrated by producing six peptides with three disulfide bonds related to four KTx sub-families and achieving peptide yields of 12-22mg per liter of culture. The developed approach can be of general use for low-cost production of various KTx, as well as other disulfide-rich peptides and proteins.

  11. Amino acid consumption in naïve and recombinant CHO cell cultures: producers of a monoclonal antibody.

    PubMed

    Carrillo-Cocom, L M; Genel-Rey, T; Araíz-Hernández, D; López-Pacheco, F; López-Meza, J; Rocha-Pizaña, M R; Ramírez-Medrano, A; Alvarez, M M

    2015-10-01

    Most commercial media for mammalian cell culture are designed to satisfy the amino acid requirements for cell growth, but not necessarily those for recombinant protein production. In this study, we analyze the amino acid consumption pattern in naïve and recombinant Chinese hamster ovary (CHO) cell cultures. The recombinant model we chose was a CHO-S cell line engineered to produce a monoclonal antibody. We report the cell concentration, product concentration, and amino acid concentration profiles in naïve and recombinant cell cultures growing in CD OptiCHO™ medium with or without amino acid supplementation with a commercial supplement (CHO CD EfficientFeed™ B). We quantify and discuss the amino acid demands due to cell growth and recombinant protein production during long term fed batch cultivation protocols. We confirmed that a group of five amino acids, constituting the highest mass fraction of the product, shows the highest depletion rates and could become limiting for product expression. In our experiments, alanine, a non-important mass constituent of the product, is in high demand during recombinant protein production. Evaluation of specific amino acid demands could be of great help in the design of feeding/supplementation strategies for recombinant mammalian cell cultures.

  12. Screening of Microorganisms Producing Cold-Active Oxidoreductases to Be Applied in Enantioselective Alcohol Oxidation. An Antarctic Survey

    PubMed Central

    Araújo, Lidiane S.; Kagohara, Edna; Garcia, Thaís P.; Pellizari, Vivian H.; Andrade, Leandro H.

    2011-01-01

    Several microorganisms were isolated from soil/sediment samples of Antarctic Peninsula. The enrichment technique using (RS)-1-(phenyl)ethanol as a carbon source allowed us to isolate 232 psychrophile/psychrotroph microorganisms. We also evaluated the enzyme activity (oxidoreductases) for enantioselective oxidation reactions, by using derivatives of (RS)-1-(phenyl)ethanol as substrates. Among the studied microorganisms, 15 psychrophile/psychrotroph strains contain oxidoreductases that catalyze the (S)-enantiomer oxidation from racemic alcohols to their corresponding ketones. Among the identified microorganisms, Flavobacterium sp. and Arthrobacter sp. showed excellent enzymatic activity. These new bacteria strains were selected for optimization study, in which the (RS)-1-(4-methyl-phenyl)ethanol oxidation was evaluated in several reaction conditions. From these studies, it was observed that Flavobacterium sp. has an excellent enzymatic activity at 10 °C and Arthrobacter sp. at 15 and 25 °C. We have also determined the growth curves of these bacteria, and both strains showed optimum growth at 25 °C, indicating that these bacteria are psychrotroph. PMID:21673897

  13. Screening of microorganisms producing cold-active oxidoreductases to be applied in enantioselective alcohol oxidation. An Antarctic survey.

    PubMed

    Araújo, Lidiane S; Kagohara, Edna; Garcia, Thaís P; Pellizari, Vivian H; Andrade, Leandro H

    2011-01-01

    Several microorganisms were isolated from soil/sediment samples of Antarctic Peninsula. The enrichment technique using (RS)-1-(phenyl)ethanol as a carbon source allowed us to isolate 232 psychrophile/psychrotroph microorganisms. We also evaluated the enzyme activity (oxidoreductases) for enantioselective oxidation reactions, by using derivatives of (RS)-1-(phenyl)ethanol as substrates. Among the studied microorganisms, 15 psychrophile/psychrotroph strains contain oxidoreductases that catalyze the (S)-enantiomer oxidation from racemic alcohols to their corresponding ketones. Among the identified microorganisms, Flavobacterium sp. and Arthrobacter sp. showed excellent enzymatic activity. These new bacteria strains were selected for optimization study, in which the (RS)-1-(4-methyl-phenyl)ethanol oxidation was evaluated in several reaction conditions. From these studies, it was observed that Flavobacterium sp. has an excellent enzymatic activity at 10 °C and Arthrobacter sp. at 15 and 25 °C. We have also determined the growth curves of these bacteria, and both strains showed optimum growth at 25 °C, indicating that these bacteria are psychrotroph.

  14. Recombinant proteinase 3 produced in different expression systems: recognition by anti-PR3 antibodies.

    PubMed

    van der Geld, Y M; Oost-Kort, W; Limburg, P C; Specks, U; Kallenberg, C G

    2000-10-20

    Anti-neutrophil cytoplasm autoantibodies (ANCA) directed against proteinase 3 (PR3) are highly sensitive and specific markers for Wegener's granulomatosis (WG). Consequently, antigen-specific assays for detection of PR3-ANCA are helpful for the diagnosis and follow-up of patients with WG. Purification of PR3 is laborious and requires large amounts of granulocytes. Therefore, several attempts have been made to produce recombinant PR3 that is recognized by PR3-ANCA. The purpose of this study was to compare the recognition of different recombinant forms of PR3 (rPR3) by anti-PR3 antibodies. Recombinant PR3 produced in E. coli (rcPR3), P. pastoris (rpPR3), insect cells using the baculovirus system (rbPR3), the human mast cell line, HMC-1 (HMC-1/PR3-S176A), or the human epithelial cell line, 293 (Delta-rPR3-S176A) as well as purified neutrophil PR3 (nPR3) were used. Recognition of these rPR3s by anti-PR3 antibodies was determined by direct and capture ELISA with 19 PR3-ANCA sera, 13 anti-PR3 mAbs and a rabbit serum raised against human PR3. In the capture ELISA rabbit anti-PR3 strongly bound nPR3 and all rPR3 products. By capture ELISA rcPR3 and rpPR3 were recognized by 11 (57%) and 13 (68%) of the 19 PR3-ANCA sera, respectively, whereas rbPR3, HMC-1/PR3-S176A, Delta-rPR3-S176A and nPR3 were recognized by all PR3-ANCA sera. By direct ELISA rabbit anti-PR3 strongly bound nPR3 and all tested rPR3 products. Using the direct ELISA none of the PR3-ANCA sera recognized rcPR3, whereas rpPR3 and rbPR3 were recognized by two (11%) and 17 (89%) of the 19 PR3-ANCA sera, respectively. All 13 anti-PR3 mAbs recognized nPR3 in the direct as well as in the capture ELISA. The rcPR3 was recognized by two mAbs in the capture ELISA but by none of the mAbs in the direct ELISA. The rpPR3 was recognized by seven mAbs in the capture ELISA and only by two mAbs in the direct ELISA. All but one of the anti-PR3 mAbs recognized rbPR3, whereas HMC-1/PR3-S176A and Delta-rPR3-S176A were recognized by

  15. Recombination produces coherent bacterial species clusters in both core and accessory genomes

    PubMed Central

    Croucher, Nicholas J.; Gutmann, Michael U.; Corander, Jukka; Hanage, William P.

    2015-01-01

    Background: Population samples show bacterial genomes can be divided into a core of ubiquitous genes and accessory genes that are present in a fraction of isolates. The ecological significance of this variation in gene content remains unclear. However, microbiologists agree that a bacterial species should be ‘genomically coherent’, even though there is no consensus on how this should be determined. Results: We use a parsimonious model combining diversification in both the core and accessory genome, including mutation, homologous recombination (HR) and horizontal gene transfer (HGT) introducing new loci, to produce a population of interacting clusters of strains with varying genome content. New loci introduced by HGT may then be transferred on by HR. The model fits well to a systematic population sample of 616 pneumococcal genomes, capturing the major features of the population structure with parameter values that agree well with empirical estimates. Conclusions: The model does not include explicit selection on individual genes, suggesting that crude comparisons of gene content may be a poor predictor of ecological function. We identify a clearly divergent subpopulation of pneumococci that are inconsistent with the model and may be considered genomically incoherent with the rest of the population. These strains have a distinct disease tropism and may be rationally defined as a separate species. We also find deviations from the model that may be explained by recent population bottlenecks or spatial structure.

  16. 2-Hydroxyglutarate produced by neomorphic IDH mutations suppresses homologous recombination and induces PARP inhibitor sensitivity

    PubMed Central

    Sulkowski, Parker L.; Corso, Christopher D.; Robinson, Nathaniel D.; Scanlon, Susan E.; Purshouse, Karin R.; Bai, Hanwen; Liu, Yanfeng; Sundaram, Ranjini K.; Hegan, Denise C.; Fons, Nathan R.; Breuer, Gregory A.; Song, Yuanbin; Mishra-Gorur, Ketu; De Feyter, Henk; de Graaf, Robin A.; Surovtseva, Yulia V.; Kachman, Maureen; Halene, Stephanie; Günel, Murat; Glazer, Peter M.; Bindra, Ranjit S.

    2017-01-01

    2-Hydroxyglutarate (2HG) exists as two enantiomers, (R)-2HG and (S)-2HG, and both are implicated in tumor progression via their inhibitory effects on α-ketoglutarate (αKG)-dependent dioxygenases. The former is an oncometabolite that is induced by the neomorphic activity conferred by isocitrate dehydrogenase-1 and -2 (IDH1/2) mutations, whereas the latter is produced under pathologic processes such as hypoxia. Here, we report that IDH1/2 mutations induce a homologous recombination (HR) defect that renders tumor cells exquisitely sensitive to poly (ADP-ribose) polymerase (PARP) inhibitors. This “BRCAness” phenotype of IDH mutant cells can be completely reversed by treatment with small molecule inhibitors of the mutant IDH1 enzyme, and, conversely, it can be entirely recapitulated by treatment with either 2HG enantiomer alone in cells with intact IDH1/2 proteins. We demonstrate IDH1-dependent PARP inhibitor sensitivity in a range of clinically relevant models, including primary patient-derived glioma cells in culture and genetically matched tumor xenografts in vivo. These findings provide the basis for a possible therapeutic strategy exploiting the biological consequences of mutant IDH, rather than attempting to block 2HG production, by targeting the 2HG-dependent HR-deficiency with PARP inhibition. Furthermore, our results uncover an unexpected link between oncometabolites, altered DNA repair, and genetic instability. PMID:28148839

  17. Spider Silk Fibers Spun from Soluble Recombinant Silk Produced in Mammalian Cells

    NASA Astrophysics Data System (ADS)

    Lazaris, Anthoula; Arcidiacono, Steven; Huang, Yue; Zhou, Jiang-Feng; Duguay, François; Chretien, Nathalie; Welsh, Elizabeth A.; Soares, Jason W.; Karatzas, Costas N.

    2002-01-01

    Spider silks are protein-based ``biopolymer'' filaments or threads secreted by specialized epithelial cells as concentrated soluble precursors of highly repetitive primary sequences. Spider dragline silk is a flexible, lightweight fiber of extraordinary strength and toughness comparable to that of synthetic high-performance fibers. We sought to ``biomimic'' the process of spider silk production by expressing in mammalian cells the dragline silk genes (ADF-3/MaSpII and MaSpI) of two spider species. We produced soluble recombinant (rc)-dragline silk proteins with molecular masses of 60 to 140 kilodaltons. We demonstrated the wet spinning of silk monofilaments spun from a concentrated aqueous solution of soluble rc-spider silk protein (ADF-3; 60 kilodaltons) under modest shear and coagulation conditions. The spun fibers were water insoluble with a fine diameter (10 to 40 micrometers) and exhibited toughness and modulus values comparable to those of native dragline silks but with lower tenacity. Dope solutions with rc-silk protein concentrations >20% and postspinning draw were necessary to achieve improved mechanical properties of the spun fibers. Fiber properties correlated with finer fiber diameter and increased birefringence.

  18. Cytotoxic and Apoptotic Effects of Recombinant Subtilase Cytotoxin Variants of Shiga Toxin-Producing Escherichia coli

    PubMed Central

    Funk, J.; Biber, N.; Schneider, M.; Hauser, E.; Enzenmüller, S.; Förtsch, C.

    2015-01-01

    In this study, the cytotoxicity of the recently described subtilase variant SubAB2-2 of Shiga toxin-producing Escherichia coli was determined and compared to the plasmid-encoded SubAB1 and the chromosome-encoded SubAB2-1 variant. The genes for the respective enzymatic active (A) subunits and binding (B) subunits of the subtilase toxins were amplified and cloned. The recombinant toxin subunits were expressed and purified. Their cytotoxicity on Vero cells was measured for the single A and B subunits, as well as for mixtures of both, to analyze whether hybrids with toxic activity can be identified. The results demonstrated that all three SubAB variants are toxic for Vero cells. However, the values for the 50% cytotoxic dose (CD50) differ for the individual variants. Highest cytotoxicity was shown for SubAB1. Moreover, hybrids of subunits from different subtilase toxins can be obtained which cause substantial cytotoxicity to Vero cells after mixing the A and B subunits prior to application to the cells, which is characteristic for binary toxins. Furthermore, higher concentrations of the enzymatic subunit SubA1 exhibited cytotoxic effects in the absence of the respective B1 subunit. A more detailed investigation in the human HeLa cell line revealed that SubA1 alone induced apoptosis, while the B1 subunit alone did not induce cell death. PMID:25824835

  19. CHO-gmt5, a novel CHO glycosylation mutant for producing afucosylated and asialylated recombinant antibodies.

    PubMed

    Haryadi, Ryan; Zhang, Peiqing; Chan, Kah Fai; Song, Zhiwei

    2013-01-01

    Engineered zinc-finger nucleases (ZFNs) are powerful tools for creating double-stranded-breaks (DSBs) in genomic DNA in a site-specific manner. These DSBs generated by ZFNs can be repaired by homology-directed repair or nonhomologous end joining, in which the latter can be exploited to generate insertion or deletion mutants. Based on published literature, we designed a pair of zinc-finger nucleases and inactivated the GDP-fucose transporter gene (Slc35c1) in a previously reported CHO mutant that has a dysfunctional CMP-sialic acid transporter gene (Slc35a1). The resulting mutant cell line, CHO-gmt5, lacks functional GDP-fucose transporter and CMP-sialic acid transporter. As a result, these cells can only produce asialylated and afucosylated glycoproteins. It is now widely recognized that removal of the core fucose from the N-glycans attached to Asn(297) of human IgG1 significantly enhances its binding to its receptor, FcγRIIIa, and thereby dramatically improves antibody-dependent cellular cytotoxicity (ADCC). Recent reports showed that removal of sialic acid from IgG1 also enhances ADCC. Therefore, CHO-gmt5 may represent a more advantageous cell line for the production of recombinant antibodies with enhanced ADCC. These cells show comparable growth rate to wild type CHO-K1 cells and uncompromised transfection efficiency, which make them desirable for use as a production line.

  20. Detection and typing of herpes simplex viruses by using recombinant immunoglobulin fragments produced in bacteria.

    PubMed Central

    Cattani, P; Rossolini, G M; Cresti, S; Santangelo, R; Burton, D R; Williamson, R A; Sanna, P P; Fadda, G

    1997-01-01

    Thirty-seven bacterial clones producing human recombinant monoclonal antibody Fab fragments (rFabs) reactive to herpes simplex virus (HSV) antigens were selected from a human combinatorial antibody library constructed in a phage-display vector by a panning procedure against an HSV lysate. Thirty-four of the HSV-specific rFabs were able to specifically recognize HSV-infected cells in indirect immunofluorescence (IF) assays; of these, 25 recognized cells infected by either HSV type 1 (HSV-1) or HSV-2, while 9 recognized only HSV-1-infected cells. One HSV type-common rFab (rFab H37) and one HSV-1-specific rFab (rFab H85) were further evaluated as reagents for viral detection and typing by IF staining in 134 HSV-positive (72 HSV-1 and 62 HSV-2) viral cultures from clinical specimens. The results obtained with these two rFabs were fully consistent with those obtained with a commercial preparation of fluorescein-labeled anti-HSV type-specific murine monoclonal antibodies. The detection sensitivity with the type-common rFab in indirect IF assays was higher overall than that provided by the type-specific murine monoclonal antibodies. Preparations of rFabs suitable for IF staining can be easily and inexpensively obtained in a clinical microbiology laboratory from Escherichia coli cultures. Similar HSV-specific rFabs, therefore, could be advantageous for in vitro diagnostic purposes. PMID:9163470

  1. Treatment of Burn and Surgical Wounds With Recombinant Human Tropoelastin Produces New Elastin Fibers in Scars.

    PubMed

    Xie, Hua; Lucchesi, Lisa; Zheng, Bo; Ladich, Elena; Pineda, Teresa; Merten, Rose; Gregory, Cynthia; Rutten, Michael; Gregory, Kenton

    2017-02-15

    Tropoelastin (TE), the soluble precursor of insoluble elastin fibers, is produced in minimal amounts in adults. Burn injuries result in inflexible collagen-rich scars because of lack of elastin fiber formation. We studied the feasibility of using recombinant human tropoelastin to enable elastin fiber production in burn and surgical scars to improve skin flexibility. In a swine hypertrophic burn scar model, normal skin and 3 × 3-cm partial thickness thermal burns underwent dermatome resection at 1 week post burn and randomized to four subcutaneous injections of saline or TE (either 0.5, 5, or 10 mg/ml) spaced 3 days apart. Two burn sites received TE injections after wound closure (0.5 or 10 mg/ml). At 90 days, skin hardness, flexibility, and histology were evaluated. All injury sites developed hypertrophic scars. New elastin fibers were found in burn scars in all injuries injected after skin closure with low (5/5) and high (6/6) TE doses (P < .05). No elastin fibers were observed without TE treatment. No significant differences in skin hardness, flexibility, or inflammation were observed. This is the first report demonstrating that subcutaneous injections of TE into surgical and burn injuries can safely produce new elastin fibers in scars. Despite the development of new elastin fibers, skin flexibility was not improved, possibly because of insufficient elastin fiber maturation or the hypertrophic model used. The ability to restore elastin fiber formation in adult skin after burns, trauma, and surgery may improve skin regeneration and reduce disabling complications of scar formation.

  2. Chaperokine function of recombinant Hsp72 produced in insect cells using a baculovirus expression system is retained.

    PubMed

    Zheng, Hongying; Nagaraja, Ganachari M; Kaur, Punit; Asea, Edwina E; Asea, Alexzander

    2010-01-01

    Extracellular heat shock protein 72 (Hsp72; inducible form of the 70-kDa heat shock protein) plays a critical role in innate and adaptive immune responses and has shown promise as an ideal adjuvant for the optimization of antigen-specific anti-tumor vaccines. Recent studies suggest that to correctly elucidate the mechanisms by which Hsp72 exerts its beneficial effects in vitro, great care must be taken to ensure that endotoxin by-products do not invalidate the findings. In this study, we have taken advantage of the baculovirus expression vector system for production of endotoxin-free recombinant Hsp72. The coding sequence of human hsp72 was recombined into the baculovirus immediately downstream of the strong polyhedron gene promoter. Ninety-six h post-infection of Sf9 insect cells with recombinant baculovirus, maximal levels of Hsp72 protein were detected. The recombinant human Hsp72 was purified by affinity chromatography from insect cells, and purity was confirmed by SDS-PAGE and mass spectrometry. The purified human recombinant Hsp72(bv) (Hsp72 produced using the BEVS) was demonstrated to have no endotoxin contamination and was shown to have stimulated potent calcium flux in the human monocytic cell line. Furthermore, recombinant Hsp72(bv) enhanced the tolerance of neuroblastoma cells to heat stress-induced cell death and displayed classical chaperokine functions including augmentation of inflammatory cytokine productions in mouse splenocytes. The production of functional, endotoxin-free recombinant human Hsp72(bv) in insect cells is inexpensive and convenient and eliminates the need of special procedures for endotoxin depletion. Endotoxin-free recombinant human Hsp72(bv) can now be used to unlock the important role Hsp72 plays in modulating immune function.

  3. Receptor specificity and functional comparison of recombinant sea bass (Dicentrarchus labrax) gonadotropins (FSH and LH) produced in different host systems.

    PubMed

    Molés, Gregorio; Zanuy, Silvia; Muñoz, Iciar; Crespo, Berta; Martínez, Iago; Mañanós, Evaristo; Gómez, Ana

    2011-06-01

    Different yields, biopotency, and in vivo pharmacokinetics are obtained for recombinant sea bass gonadoltropins depending on the production system and DNA construct, but they show specific activation of their corresponding receptors. Gonadotropins (GTHs) are glycoprotein hormones that play a major role in the regulation of gonadal functions. Recently, we succeeded in isolating the native sea bass Fsh from sea bass pituitaries, but to ensure the availability of bioactive GTHs and no cross-contamination with other related glycoproteins, recombinant sea bass GTHs were produced using two expression systems-insect and mammalian cells-and different constructs that yielded tethered or noncovalently bound dimers. Their production levels, binding specificity to their homologous cognate receptors, and bioactivity were investigated and compared. Both expression systems were successful in the generation of bioactive recombinant GTHs, but insect Sf9 cells yielded higher amounts of recombinant proteins than mammalian Chinese Hamster Ovary (CHO) stable clones. All recombinant GTHs activated their cognate receptors without cross-ligand binding and were able to stimulate sea bass gonadal steroidogenesis in vitro, although with different biopotencies. To assess their use for in vivo applications, their half-life in sea bass plasma was evaluated. Sf9-GTHs had a lower in vivo stability compared with CHO-GTHs due to their rapid clearance from the blood circulation. Cell-dependent glycosylation could be contributing to the final in vivo stability and biopotency of these recombinant glycoproteins. In conclusion, both insect and mammalian expression systems produced bioactive sea bass recombinant gonadotropins, although with particular features useful for different proposes (e.g., antibody production or in vivo studies, respectively).

  4. A Gateway recombination herpesvirus cloning system with negative selection that produces vectorless progeny.

    PubMed

    Kunec, Dusan; van Haren, Sandra; Burgess, Shane C; Hanson, Larry A

    2009-01-01

    Crossover recombination based on the lambda phage integration/excision functions enables insertion of a gene of interest into a specific locus by a simple one-step in vitro recombination reaction. Recently, a highly efficient recombination system for targeted mutagenesis, which utilizes lambda phage crossover recombination cloning, has been described for a human herpesvirus 2 bacterial artificial chromosome (BAC). The disadvantages of the system are that it allows only neutral selection (loss of green fluorescent protein) of desired recombinants and that it regenerates herpesvirus progeny containing the BAC sequence inserted in the herpesvirus genome. In this study, the existing channel catfish herpesvirus (CCV) infectious clone (in the form of overlapping fragments) was modified to allow introduction of foreign genes by modified lambda phage crossover recombination cloning. This novel system enables negative and neutral selection and regenerates vectorless herpesvirus progeny. Construction of two CCV mutants expressing lacZ, one from the native CCV ORF5 promoter and the other from the immediate-early cytomegalovirus promoter, demonstrated the efficiency and reliability of this system. This novel cloning system enables rapid incorporation, direct delivery and high-level expression of foreign genes by a herpesvirus. This system has broad utility and could be used to facilitate development of recombinant viruses, viral vectors and better vaccines.

  5. Challenges in electrochemical pre-purification of recombinant proteins from green plant tissues: sgfp produced in tobacco leaves.

    PubMed

    Robić, Goran

    2013-01-01

    The use of recombinant proteins has increased greatly in recent years, as have the number of techniques and materials used for their production and purification. The principal advantage of using plants as bioreactors is the cost of the recombinant protein production, which is about 1000-fold lower as in the case of using CHO cells commonly applied in industry today. Among the different types of "green" bioreactors being studied today, there is a general consensus among scientists that production in green plant tissues such as leaves is more feasible. However, the presence of chlorophyll and phenolic compounds in plant extracts, which can precipitate and denature the proteins besides damaging separation membranes and gels, makes this technology impracticable on a commercial scale. Electrochemically produced aluminium hydroxide gel can be used to adsorb these compounds, and pre-purify recombinant synthetic green fluorescent protein (sGFP) produced in Nicotiana benthamiana leaves. Removal efficiencies of 99.7% of chlorophyll, 88.5% of phenolic compounds, and 38.5% of native proteins from the N. benthamiana extracts were achieved without removing sGFP from the extracts. Since electrochemical preparation of aluminum hydroxide gel is a cost-effective technique, its use can substantially contribute to the development of future production platforms for recombinant proteins produced in green plant tissues of pharmaceutical and industrial interest.

  6. GATEWAY technology and E. coli recombinant system produce a properly folded and functional recombinant allergen of the lipid transfer protein of apple (Mal d 3).

    PubMed

    Borges, Jean-Philippe; Culerrier, Raphaël; Aldon, Didier; Barre, Annick; Benoist, Hervé; Saurel, Olivier; Milon, Alain; Didier, Alain; Rougé, Pierre

    2010-04-01

    The lipid transfer protein of apple fruit, Mal d 3, has been produced as a soluble recombinant protein in transformed Escherichia coli cells using the GATEWAY technology. Circular dichroism spectra showing the protein essentially consists of alpha-helices indicate that the rMal d 3 is properly folded. The (1)H NMR spectra also indicates a correct fold for the recombinant allergen. The reactivity of rMal d 3 towards IgE from apple allergic patients and in vitro degranulation activity measured on transformed rat basophil leukemia cells expressing the human Fc epsilon RI alpha-subunit of rMal d 3 is similar to those of the native allergen purified from apple fruits. The expression of active rMal d 3 in E. coli is readily feasible and offers an interesting alternative to the production of recombinant allergen in the yeast Pichia pastoris. This expression in E. coli open the way to the modification of Mal d 3 by site-directed mutagenesis for immunotherapy purposes. (c) 2009 Elsevier Inc. All rights reserved.

  7. Production of specific IgY antibody to the recombinant FanC protein produced in Escherichia coli

    PubMed Central

    Nasiri, Khadijeh; Zibaee, Saeed; Nassiri, Mohammadreza; Tahmoorespur, Mojtaba; Haghparast, Alireza

    2016-01-01

    Objective(s): Enterotoxigenic Escherichia coli (ETEC) strains are one of the primary causes of diarrhea in newborn calves and in humans, pigs, and sheep. IgY technology has been identified as a promising alternative to generating a mass amount of specific antibody for use in immunotherapy and immunodiagnostics. The purpose of this study was to produce specific antibody by egg yolk antibody (IgY) to recombinant FanC protein from ETEC. Materials and Methods: FanC (K99) gene was amplified from ETEC by specific primers and polymerase chain reaction. The gene was cloned and subcloned into pTZ57R/T and pET32a (+) vectors, respectively. Recombinant vector was transferred into E. coli BL21 CodonPlus (DE3). Protein expression was investigated by 1 mM IPTG induction. Hens were immunized by the purified recombinant FanC protein. The activity and specificity of the IgY antibody were detected by dot-blotting, Western blotting, and indirect ELISA. Results: We obtained FanC specific IgYs by immunizing the hens with the recombinant FanC protein. The anti-FanC IgY showed binding specifically to the FanC protein of ETEC. Conclusion: The results emphasize that specific IgY against the recombinant FanC protein could be recommended as a candidate for passive immunization against ETEC infection in animals and humans. PMID:27746871

  8. Study of the micro-organisms associated with the fermented bread (khamir) produced from sorghum in Gizan region, Saudi Arabia.

    PubMed

    Gassem, M A

    1999-02-01

    Traditional bread (khamir) was made from sorghum flour of two local varieties, Bayadh and Hamra. The bread was prepared by mixing the sorghum flour with water and spices (onion, garlic, lemon juice and fenugreek) in a 1:0.8 (w/w) ratio and fermented for 24 h at 30 degrees C. Two other fermentations were carried out using an inoculum from the previous fermentation. The micro-organisms were isolated from different plates and identified using different characterization systems. Both total bacterial populations and lactic acid bacteria increased with fermentation time and reached the highest number at 16 h (first fermentation) and at 8 h (second and third fermentation). The content of lactic acid was increased with time to reach 1.2%, but the increase was higher for the second and third fermentations (1.6% each). The pH dropped with time from 6.77 to 4.35 in the first fermentation and from 6.65 to 4.18, and 6.57-3.93, in the second and third fermentations, respectively. The microorganisms, which were isolated and characterized during the 24 h fermentation, included: bacteria (Pediococcus pentosaceus, Lactobacillus brevis, Lact. lactis subsp. lactis, Lact. cellobiosus, Klebsiella oxytoca, Kl. pneumoniae, Enterobacter aerogenes, Ent. sakazakii, Serratia marcescens and Ser. odourifera), moulds (Penicillium sp., Rhizopus sp., Aspergillus niger, Alternaria sp., Fusarium sp. and Mucor sp.) and yeasts (Candida parapsilosis, C. orvegnsis and Rhodotorula glutinis).

  9. An optimized sericin-1 expression system for mass-producing recombinant proteins in the middle silk glands of transgenic silkworms.

    PubMed

    Wang, Feng; Xu, Hanfu; Yuan, Lin; Ma, Sanyuan; Wang, Yuancheng; Duan, Xiaoli; Duan, Jianping; Xiang, Zhonghuai; Xia, Qingyou

    2013-10-01

    The middle silk gland (MSG) of silkworm is thought to be a potential host for mass-producing valuable recombinant proteins. Transgenic MSG expression systems based on the usage of promoter of sericin1 gene (sericin-1 expression system) have been established to produce various recombinant proteins in MSG. However, further modifying the activity of the sericin-1 expression system to yield higher amounts of recombinant proteins is still necessary. In this study, we provide an alternative modification strategy to construct an efficient sericin-1 expression system by using the hr3 enhancer (hr3 CQ) from a Chongqing strain of the Bombyx mori nuclear polyhedrosis virus (BmNPV) and the 3'UTRs of the fibroin heavy chain (Fib-HPA), the fibroin light chain (Fib-LPA), and Sericin1 (Ser1PA) genes. We first analyzed the effects of these DNA elements on expression of luciferase, and found that the combination of hr3 CQ and Ser1PA was most effective to increase the activity of luciferase. Then, hr3 CQ and Ser1PA were used to modify the sericin1 expression system. Transgenic silkworms bearing these modified sericin1 expression vectors were generated by a piggyBac transposon mediated genetic transformation method. Our results showed that mRNA level of DsRed reporter gene in transgenic silkworms containing hr3 CQ and Ser1PA significantly increased by 9 fold to approximately 83 % of that of endogenous sericin1. As the results of that, the production of recombinant RFP increased by 16 fold to 9.5 % (w/w) of cocoon shell weight. We conclude that this modified sericin-1 expression system is efficient and will contribute to the MSG as host to mass produce valuable recombinant proteins.

  10. Exploiting translational coupling for the selection of cells producing toxic recombinant proteins from expression vectors.

    PubMed

    Tagliavia, Marcello; Cuttitta, Angela

    2016-01-01

    High rates of plasmid instability are associated with the use of some expression vectors in Escherichia coli, resulting in the loss of recombinant protein expression. This is due to sequence alterations in vector promoter elements caused by the background expression of the cloned gene, which leads to the selection of fast-growing, plasmid-containing cells that do not express the target protein. This phenomenon, which is worsened when expressing toxic proteins, results in preparations containing very little or no recombinant protein, or even in clone loss; however, no methods to prevent loss of recombinant protein expression are currently available. We have exploited the phenomenon of translational coupling, a mechanism of prokaryotic gene expression regulation, in order to select cells containing plasmids still able to express recombinant proteins. Here we designed an expression vector in which the cloned gene and selection marker are co-expressed. Our approach allowed for the selection of the recombinant protein-expressing cells and proved effective even for clones encoding toxic proteins.

  11. Microorganism immobilization

    DOEpatents

    Compere, Alicia L.; Griffith, William L.

    1981-01-01

    Live metabolically active microorganisms are immobilized on a solid support by contacting particles of aggregate material with a water dispersible polyelectrolyte such as gelatin, crosslinking the polyelectrolyte by reacting it with a crosslinking agent such as glutaraldehyde to provide a crosslinked coating on the particles of aggregate material, contacting the coated particles with live microorganisms and incubating the microorganisms in contact with the crosslinked coating to provide a coating of metabolically active microorganisms. The immobilized microorganisms have continued growth and reproduction functions.

  12. A Recombinant Fungal Chitin Deacetylase Produces Fully Defined Chitosan Oligomers with Novel Patterns of Acetylation

    PubMed Central

    Naqvi, Shoa; Cord-Landwehr, Stefan; Singh, Ratna; Bernard, Frank; Kolkenbrock, Stephan; El Gueddari, Nour Eddine

    2016-01-01

    . tritici in the periplasm of E. coli as a fusion protein with the maltose-binding protein; this strategy allows the production of these difficult-to-express enzymes in sufficient quantities for them to be characterized and optimized through protein engineering. Here, the recombinant enzyme was used to produce partially acetylated chitosan oligosaccharides from chitin oligomers, whereby the pronounced regioselectivity of the enzyme led to the production of defined products with novel patterns of acetylation. This approach widens the scope for both the production and functional analysis of chitosan oligomers and thus will eventually allow the detailed molecular structure-function relationships of biologically active chitosans to be studied, which is essential for developing applications for these functional biopolymers for a circular bioeconomy, e.g., in agriculture, medicine, cosmetics, and food sciences. PMID:27590819

  13. Haematococcus as a promising cell factory to produce recombinant pharmaceutical proteins.

    PubMed

    Saei, Amir Ata; Ghanbari, Parisa; Barzegari, Abolfazl

    2012-11-01

    The need for recombinant pharmaceutical proteins has urged scientists all over the world to search for better protein expression systems which have higher capabilities and flexibilities. Although a number of protein expression systems are now available, no system is ideal and different systems lack specific properties. Here, microalga Haematococcus is discussed as a new protein expression system which merits cheap growth medium, fast growth rate, ease of manipulation and scale-up, ease of transformation, potential of exploiting in bioreactors and ability to exert post-translational modifications to the proteins. This green single-cell plant has favorable biological and biotechnological features for production of remarkable yields of recombinant proteins with high functionality. In this review article, we highlight the favorable biotechnological characteristics of Haematococcus for lowering costs and facilitating scale-up of recombinant protein production along with its superior biological features for genetic engineering.

  14. Detection of recombinant human lactoferrin and lysozyme produced in a bitransgenic cow.

    PubMed

    Kaiser, Germán G; Mucci, Nicolás C; González, Vega; Sánchez, Lourdes; Parrón, José A; Pérez, María D; Calvo, Miguel; Aller, Juan F; Hozbor, Federico A; Mutto, Adrián A

    2017-03-01

    Lactoferrin and lysozyme are 2 glycoproteins with great antimicrobial activity, being part of the nonspecific defensive system of human milk, though their use in commercial products is difficult because human milk is a limited source. Therefore, many investigations have been carried out to produce those proteins in biological systems, such as bacteria, yeasts, or plants. Mammals seem to be more suitable as expression systems for human proteins, however, especially for those that are glycosylated. In the present study, we developed a bicistronic commercial vector containing a goat β-casein promoter and an internal ribosome entry site fragment between the human lactoferrin and human lysozyme genes to allow the introduction of both genes into bovine adult fibroblasts in a single transfection. Embryos were obtained by somatic cell nuclear transfer, and, after 6 transferences to recipients, 3 pregnancies and 1 viable bitransgenic calf were obtained. The presence of the vector was confirmed by fluorescent in situ hybridization of skin cells. At 13 mo of life and after artificial induction of lactation, both recombinant proteins were found in the colostrum and milk of the bitransgenic calf. Human lactoferrin concentration in the colostrum was 0.0098 mg/mL and that in milk was 0.011 mg/mL; human lysozyme concentration in the colostrum was 0.0022 mg/mL and that in milk was 0.0024 mg/mL. The molar concentration of both human proteins revealed no differences in protein production of the internal ribosome entry site upstream and downstream protein. The enzymatic activity of lysozyme in the transgenic milk was comparable to that of human milk, being 6 and 10 times higher than that of bovine lysozyme present in milk. This work represents an important step to obtain multiple proteins or enhance single protein production by using animal pharming and fewer regulatory and antibiotic-resistant foreign sequences, allowing the design of humanized milk with added biological value for

  15. Characterization and biological activities of recombinant human plasminogen kringle 1-3 produced in Escherichia coli.

    PubMed

    You, Weon-Kyoo; So, Seung-Ho; Sohn, Young-Doug; Lee, Hyosil; Park, Doo-Hong; Chung, Soo-Il; Chung, Kwang-Hoe

    2004-07-01

    Angiogenesis, the formation of new capillaries from preexisting blood vessels, is involved in many pathological conditions, for example, tumorigenesis, diabetic retinopathy, and rheumatoid arthritis. Angiostatin, which contains the kringle 1-4 domains of plasminogen, is known to be a potent inhibitor of angiogenesis and a strong suppressor of various solid tumors. In this study, we expressed recombinant protein containing the kringle 1-3 domains of human plasminogen in Escherichia coli and investigated its biological activities. The protein was successfully refolded from inclusion bodies and purified at a 30% overall yield, as a single peak by HPLC. The purified recombinant protein had biochemical properties that were similar to those of the native form, which included molecular size, lysine-binding capacity, and immunoreactivity with a specific antibody. The recombinant protein was also found to strongly inhibit the proliferation of bovine capillary endothelial cells in vitro, and the formation of new capillaries on chick embryos. In addition, it suppressed the growth of primary Lewis lung carcinoma and B16 melanoma in an in vivo mouse model. Our findings suggest that the recombinant kringle 1-3 domains in a prokaryote expression system have anti-angiogenic activities, which may be useful in clinical and basic research in the field of angiogenesis.

  16. Optimization of culturing conditions of recombined Escherichia coli to produce umami octopeptide-containing protein.

    PubMed

    Zhang, Yin; Wei, Xiong; Lu, Zhou; Pan, Zhongli; Gou, Xinhua; Venkitasamy, Chandrasekar; Guo, Siya; Zhao, Liming

    2017-07-15

    Using synthesized peptides to verify the taste of natural peptides was probably the leading cause for tasting disputes regarding umami peptides. A novel method was developed to prepare the natural peptide which could be used to verify the taste of umami peptide. A controversial octopeptide was selected and gene engineering was used to structure its Escherichia coli. expressing vector. A response surface method was adopted to optimize the expression conditions of the recombinant protein. The results of SDS-PAGE for the recombinant protein indicated that the recombinant expression system was successfully structured. The fitting results of the response surface experiment showed that the OD600 value was the key factor which influenced the expression of the recombinant protein. The optimal culturing process conditions predicted with the fitting model were an OD600 value of 0.5, an IPTG concentration of 0.6mM, a culturing temperature of 28.75°C and a culturing time of 5h.

  17. The recombinant EHV-1 vector producing CDV hemagglutinin as potential vaccine against canine distemper.

    PubMed

    Pan, Zihao; Liu, Jin; Ma, Jiale; Jin, Qiuli; Yao, Huochun; Osterrieder, Nikolaus

    2017-10-01

    Canine distemper virus (CDV), is a pantropic agent of morbillivirus that causes fetal disease in dogs. Base on a broad host rang of CDV, the continued vaccines inoculation is unavoidable to pose gene recombination risk in vaccine virus and wild virus. The current study presents the construction of novel vectors, using equine herpesvirus type 1 (EHV-1) expressing the canine distemper virus (CDV). The recent field strain hemagglutinin protein and nucleoprotein were used for the construction of the viral vector vaccines. Based on the Bacterial artificial chromosome (BAC) genomes of EHV-1 RacH strain, the recombinant EHV-1 vaccine virus encoding CDV hemagglutinin protein (EHV-H) or CDV nucleoprotein (EHV-N) was constructed separately. The constructed BACs were rescued after 72 h post infection, and the expression of H or N in the recombinant viruses was confirmed by western-blotting. Furthermore, high levels of neutralizing antibodies were induced persistently following vaccination in the groups EHV-H&EHV-N and EHV-H, but the EHV-N group. The groups of vaccinated EHV-H and EHV-H&EHV-N pups were monitored for clinical signs, whereas the vaccinated EHV-N group developed moderate symptoms. The present study demonstrated that EHV-1 based recombinant virus carrying CDV H could be a promising vaccine candidate against canine distemper. Copyright © 2017. Published by Elsevier Ltd.

  18. Native-sized recombinant spider silk protein produced in metabolically engineered Escherichia coli results in a strong fiber

    PubMed Central

    Xia, Xiao-Xia; Qian, Zhi-Gang; Ki, Chang Seok; Park, Young Hwan; Kaplan, David L.; Lee, Sang Yup

    2010-01-01

    Spider dragline silk is a remarkably strong fiber that makes it attractive for numerous applications. Much has thus been done to make similar fibers by biomimic spinning of recombinant dragline silk proteins. However, success is limited in part due to the inability to successfully express native-sized recombinant silk proteins (250–320 kDa). Here we show that a 284.9 kDa recombinant protein of the spider Nephila clavipes is produced and spun into a fiber displaying mechanical properties comparable to those of the native silk. The native-sized protein, predominantly rich in glycine (44.9%), was favorably expressed in metabolically engineered Escherichia coli within which the glycyl-tRNA pool was elevated. We also found that the recombinant proteins of lower molecular weight versions yielded inferior fiber properties. The results provide insight into evolution of silk protein size related to mechanical performance, and also clarify why spinning lower molecular weight proteins does not recapitulate the properties of native fibers. Furthermore, the silk expression, purification, and spinning platform established here should be useful for sustainable production of natural quality dragline silk, potentially enabling broader applications. PMID:20660779

  19. Prevalence of Extended-Spectrum Beta-Lactamases-Producing Microorganisms in Patients Admitted at KRRH, Southwestern Uganda

    PubMed Central

    Andrew, Baguma; Kagirita, Atek

    2017-01-01

    The emergence of extended-spectrum beta-lactamase- (ESBL-) producing pathogenic bacteria at Kabale Regional Referral Hospital (KRRH), located in southwestern Uganda, is of great concern: a phenomenon that worries clinicians and other healthcare workers due to the serious threat they pose to patients. This current study aimed at determining the phenotypic detection of ESBL-producing strains of E. coli, Klebsiella sp., and Proteus sp. isolated from clinical specimens and their prevalence in patients admitted at KRRH. We used combined disc diffusion technique to detect and establish the presence of ESBLs-producing bacteria. Of the 100 tested bacterial isolates, 89 (89%) were identified as ESBL-producing bacteria. Klebsiella sp. predominated in the samples (46 (52%)), presenting the highest frequency of ESBLs producing followed by E. coli (39 (44%)) and Proteus mirabilis (4 (4.5%)) from the combined disk diffusion. PMID:28270849

  20. Integrated approach to produce a recombinant, His-tagged human α-galactosidase A in mammalian cells.

    PubMed

    Corchero, José Luis; Mendoza, Rosa; Lorenzo, Julia; Rodríguez-Sureda, Victor; Domínguez, Carmen; Vázquez, Esther; Ferrer-Miralles, Neus; Villaverde, Antonio

    2011-01-01

    Successful production of recombinant proteins (r-proteins) by transient gene expression (TGE) depends on several parameters (including producer cells, culture conditions, transfection procedure, or expression vector) that should be optimized when producing any recombinant product. In this work, TGE-based production of human α-galactosidase A (GLA) is described. Producer cells, expression vectors, and parameters influencing cell metabolism after transfection have been tested. The enzyme is secreted, has the right molecular weight, and is enzymatically active. Productivities of up to 30-40 mg/L have been achieved, with a simple, fast procedure. A 6 × His tag allows enzyme purification in a single step, rendering a highly pure product. We propose a TGE-based protocol able to produce up to several milligrams per liter of highly pure, active GLA in a time as short as a few days. By this, enough amounts of engineered versions of the enzyme can be easily produced to be tested in vitro or in preclinical trials.

  1. High-efficiency homologous recombination in the oil-producing alga Nannochloropsis sp.

    PubMed Central

    Kilian, Oliver; Benemann, Christina S. E.; Niyogi, Krishna K.; Vick, Bertrand

    2011-01-01

    Algae have reemerged as potential next-generation feedstocks for biofuels, but strain improvement and progress in algal biology research have been limited by the lack of advanced molecular tools for most eukaryotic microalgae. Here we describe the development of an efficient transformation method for Nannochloropsis sp., a fast-growing, unicellular alga capable of accumulating large amounts of oil. Moreover, we provide additional evidence that Nannochloropsis is haploid, and we demonstrate that insertion of transformation constructs into the nuclear genome can occur by high-efficiency homologous recombination. As examples, we generated knockouts of the genes encoding nitrate reductase and nitrite reductase, resulting in strains that were unable to grow on nitrate and nitrate/nitrite, respectively. The application of homologous recombination in this industrially relevant alga has the potential to rapidly advance algal functional genomics and biotechnology. PMID:22123974

  2. High-efficiency homologous recombination in the oil-producing alga Nannochloropsis sp.

    PubMed

    Kilian, Oliver; Benemann, Christina S E; Niyogi, Krishna K; Vick, Bertrand

    2011-12-27

    Algae have reemerged as potential next-generation feedstocks for biofuels, but strain improvement and progress in algal biology research have been limited by the lack of advanced molecular tools for most eukaryotic microalgae. Here we describe the development of an efficient transformation method for Nannochloropsis sp., a fast-growing, unicellular alga capable of accumulating large amounts of oil. Moreover, we provide additional evidence that Nannochloropsis is haploid, and we demonstrate that insertion of transformation constructs into the nuclear genome can occur by high-efficiency homologous recombination. As examples, we generated knockouts of the genes encoding nitrate reductase and nitrite reductase, resulting in strains that were unable to grow on nitrate and nitrate/nitrite, respectively. The application of homologous recombination in this industrially relevant alga has the potential to rapidly advance algal functional genomics and biotechnology.

  3. Interactions between plants and microorganisms

    USDA-ARS?s Scientific Manuscript database

    Allelopathic microorganisms comprise rhizobacteria and fungi that colonize the surfaces of plant roots, and produce and release phytotoxic metabolites, similar to allelochemicals, that detrimentally affect growth of their host plants. The allelopathic microorganisms are grouped separately from typic...

  4. Prevalence of extended-spectrum beta-lactamases-producing microorganisms in nosocomial patients and molecular characterization of the shv type isolates

    PubMed Central

    de Oliveira, Caio Fernando; Salla, Adenilde; Lara, Valéria Maria; Rieger, Alexandre; Horta, Jorge André; Alves, Sydney Hartz

    2010-01-01

    The emergence of Extended-Spectrum Beta-Lactamase (ESBL)-producing microorganisms in Brazilian hospitals is a challenge that concerns scientists, clinicians and healthcare institutions due to the serious risk they pose to confined patients. The goal of this study was the detection of ESBL production by clinical strains of Escherichia coli and Klebsiella sp. isolated from pus, urine and blood of patients at Hospital Universitário Santa Maria, Rio Grande Sul, RS, Brazil and the genotyping of the isolates based on bla SHV genes. The ESBL study was carried out using the Combined Disc Method, while Polymerase Chain Reaction (PCR) was used to study the bla SHV genes. Of the 90 tested isolates, 55 (61.1%) were identified as ESBL-producing by the combined disk method. The bla SHV genes were found in 67.8% of these microorganisms. K. pneumoniae predominated in the samples, presenting the highest frequency of positive results from the combined disk and PCR. PMID:24031491

  5. Effective non-denaturing purification method for improving the solubility of recombinant actin-binding proteins produced by bacterial expression.

    PubMed

    Chung, Jeong Min; Lee, Sangmin; Jung, Hyun Suk

    2017-05-01

    Bacterial expression is commonly used to produce recombinant and truncated mutant eukaryotic proteins. However, heterologous protein expression may render synthesized proteins insoluble. The conventional method used to express a poorly soluble protein, which involves denaturation and refolding, is time-consuming and inefficient. There are several non-denaturing approaches that can increase the solubility of recombinant proteins that include using different bacterial cell strains, altering the time of induction, lowering the incubation temperature, and employing different detergents for purification. In this study, we compared several non-denaturing protocols to express and purify two insoluble 34 kDa actin-bundling protein mutants. The solubility of the mutant proteins was not affected by any of the approaches except for treatment with the detergent sarkosyl. These results indicate that sarkosyl can effectively improve the solubility of insoluble proteins during bacterial expression. Copyright © 2016. Published by Elsevier Inc.

  6. The pEAQ vector series: the easy and quick way to produce recombinant proteins in plants.

    PubMed

    Peyret, Hadrien; Lomonossoff, George P

    2013-09-01

    The pEAQ vectors are a series of plasmids designed to allow easy and quick production of recombinant proteins in plants. Their main feature is the use of the Cowpea Mosaic Virus hypertranslational "CPMV-HT" expression system, which provides high yields of recombinant protein through extremely high translational efficiency without the need for viral replication. Since their creation, the pEAQ vectors have been used to produce a wide variety of proteins in plants. Viral proteins and Virus-Like Particles (VLPs) have been of particular interest, but other types of proteins including active enzymes have also been expressed. While the pEAQ vectors have mostly been used in a transient expression context, through agroinfiltration of leaves, they have also been shown to be suitable for the production of stably transformed lines of both cell cultures and whole plants. This paper looks back on the genesis of the pEAQ vectors and reviews their use so far.

  7. One single method to produce native and Tat-fused recombinant human α-synuclein in Escherichia coli

    PubMed Central

    2013-01-01

    Background Human α-synuclein is a small-sized, natively unfolded protein that in fibrillar form is the primary component of Lewy bodies, the pathological hallmark of Parkinson’s disease. Experimental evidence suggests that α-synuclein aggregation is the key event that triggers neurotoxicity although additional findings have proposed a protective role of α-synuclein against oxidative stress. One way to address the mechanism of this protective action is to evaluate α-synuclein-mediated protection by delivering this protein inside cells using a chimeric protein fused with the Tat-transduction domain of HIV Tat, named TAT-α-synuclein. Results A reliable protocol was designed to efficiently express and purify two different forms of human α-synuclein. The synthetic cDNAs encoding for the native α-synuclein and the fusion protein with the transduction domain of Tat protein from HIV were overexpressed in a BL21(DE3) E. coli strain as His-tagged proteins. The recombinant proteins largely localized (≥ 85%) to the periplasmic space. By using a quick purification protocol, based on recovery of periplasmic space content and metal-chelating chromatography, the recombinant α-synuclein protein forms could be purified in a single step to ≥ 95% purity. Both α-synuclein recombinant proteins form fibrils and the TAT-α-synuclein is also cytotoxic in the micromolar concentration range. Conclusions To further characterize the molecular mechanisms of α-synuclein neurotoxicity both in vitro and in vivo and to evaluate the relevance of extracellular α-synuclein for the pathogenesis and progression of Parkinson’s disease, a suitable method to produce different high-quality forms of this pathological protein is required. Our optimized expression and purification procedure offers an easier and faster means of producing different forms (i.e., both the native and the TAT-fusion form) of soluble recombinant α-synuclein than previously described procedures. PMID:23557146

  8. Rational Engineering of Recombinant Picornavirus Capsids to Produce Safe, Protective Vaccine Antigen

    PubMed Central

    Burman, Alison; Jackson, Terry; Ren, Jingshan; Loureiro, Silvia; Jones, Ian M.; Fry, Elizabeth E.; Stuart, David I.; Charleston, Bryan

    2013-01-01

    Foot-and-mouth disease remains a major plague of livestock and outbreaks are often economically catastrophic. Current inactivated virus vaccines require expensive high containment facilities for their production and maintenance of a cold-chain for their activity. We have addressed both of these major drawbacks. Firstly we have developed methods to efficiently express recombinant empty capsids. Expression constructs aimed at lowering the levels and activity of the viral protease required for the cleavage of the capsid protein precursor were used; this enabled the synthesis of empty A-serotype capsids in eukaryotic cells at levels potentially attractive to industry using both vaccinia virus and baculovirus driven expression. Secondly we have enhanced capsid stability by incorporating a rationally designed mutation, and shown by X-ray crystallography that stabilised and wild-type empty capsids have essentially the same structure as intact virus. Cattle vaccinated with recombinant capsids showed sustained virus neutralisation titres and protection from challenge 34 weeks after immunization. This approach to vaccine antigen production has several potential advantages over current technologies by reducing production costs, eliminating the risk of infectivity and enhancing the temperature stability of the product. Similar strategies that will optimize host cell viability during expression of a foreign toxic gene and/or improve capsid stability could allow the production of safe vaccines for other pathogenic picornaviruses of humans and animals. PMID:23544011

  9. Decreased fluidity of cell membranes causes a metal ion deficiency in recombinant Saccharomyces cerevisiae producing carotenoids.

    PubMed

    Liu, Peitong; Sun, Liang; Sun, Yuxia; Shang, Fei; Yan, Guoliang

    2016-04-01

    The genome-wide transcriptional responses of S. cerevisiae to heterologous carotenoid biosynthesis were investigated using DNA microarray analysis. The results show that the genes involved in metal ion transport were specifically up-regulated in the recombinant strain, and metal ions, including Cu(2+), Fe(2+), Mn(2+), and Mg(2+), were deficient in the recombinant strain compared to the ion content of the parent strain. The decrease in metal ions was ascribed to a decrease in cell membrane (CM) fluidity caused by lower levels of unsaturated fatty acids and ergosterol. This was confirmed by the observation that metal ion levels were restored when CM fluidity was increased by supplying linoleic acid. In addition, a 24.3 % increase in the β-carotene concentration was observed. Collectively, our results suggest that heterologous production of carotenoids in S. cerevisiae can induce cellular stress by rigidifying the CM, which can lead to a deficiency in metal ions. Due to the importance of CM fluidity in cellular physiology, maintaining normal CM fluidity might be a potential approach to improving carotenoid production in genetically engineered S. cerevisiae.

  10. Immunodiagnosis of Prune dwarf virus using antiserum produced to its recombinant coat protein.

    PubMed

    Abou-Jawdah, Yusuf; Sobh, Hana; Cordahi, Nada; Kawtharani, Hadia; Nemer, George; Maxwell, Douglas P; Nakhla, Mark K

    2004-10-01

    Certification represents the first line of defense against fruit tree viruses. For certification or surveys dealing with large number of samples, ELISA is still considered the technique of choice and requires a continuous supply of good quality antibodies. Prune dwarf virus (PDV) is among the major viruses affecting stone fruits; it belongs to the genus Ilarvirus named so for its isometric labile particles. Recombinant DNA technology was investigated for production of PDV antiserum to avoid labile virus purification and virus maintenance problems. The PDV coat protein gene (CP) was cloned into a protein expression bacterial plasmid vector which allowed a good level of expression of up to 2mg native protein/L culture. The recombinant PDV CP was injected into rabbits and the crude antiserum was successfully used in indirect ELISA at dilutions of up to 1:5000 to detect PDV in infected leaf samples. Similar results were obtained in dot blot immunoassays (DBIA). The antibodies were used in double antibody sandwich enzyme-linked immunosorbent assay (DAS-ELISA) and results were comparable to a reference commercial kit. The crude antiserum was efficiently used for coating ELISA plates, thereby reducing test costs.

  11. Construction of Recombinant Bacmid Containing M2e-Ctxb and Producing the Fusion Protein in Insect Cell Lines

    PubMed Central

    Mirzaei, Nima; Mokhtari Azad, Talat; Nategh, Rakhshandeh; Soleimanjahi, Hoorieh; Amirmozafari, Nour

    2014-01-01

    Background: Sequence variations in glycoproteins of influenza virus surface impel us to design new candidate vaccines yearly. Ectodomain of influenza M2 protein is a surface and highly conserved protein. M2e in influenza vaccines may eliminate the need for changing vaccine formulation every year. Objectives: In this study, a recombinant baculovirus containing M2e and cholera toxin subunit B fusion gene was generated with transposition process to express in large amounts in insect cell lines. Materials and Methods: M2e-ctxB fusion gene was created and cloned into pFastBac HT. The recombinant vector was transformed into DH10Bac cells to introduce the fusion gene into the bacmid DNA via a site-specific transposition process. The recombinant bacmid was then extracted from white colonies and further analyzed using PCR, DNA sequence analyzing, and indirect immunofluorescence assay. Results: PCR and DNA sequence analyzing results showed that the fusion gene was constructed as a single open reading frame and was successfully inserted into bacmid DNA. Moreover, indirect immunofluorescence results showed that the fusion gene was successfully expressed. Conclusions: Baculovirus expression vector system is valuable to produce M2e based influenza vaccines due to its simple utilization and ease of target gene manipulation. The expressed protein in such systems can improve the evaluating process of new vaccination strategies. PMID:24719728

  12. Enhancing isomaltulose production by recombinant Escherichia coli producing sucrose isomerase: culture medium optimization containing agricultural wastes and cell immobilization.

    PubMed

    Li, Sha; Xu, Hong; Yu, Jianguang; Wang, Yanyuan; Feng, Xiaohai; Ouyang, Pingkai

    2013-10-01

    Isomaltulose is a structural isomer of sucrose commercially used in food industries. In this work, recombinant Escherichia coli producing sucrose isomerase (SIase) was used to convert sucrose into isomaltulose. To develop an economical industrial medium, untreated cane molasses (10.63 g l⁻¹), yeast extract (25.93 g l⁻¹), and corn steep liquor (10.45 g l⁻¹) were used as main culture compositions for SIase production. The relatively high SIase activity (14.50 ± 0.11 U mg DCW⁻¹) was obtained by the recombinant cells. To the best of our knowledge, this is the first investigation on SIase production by engineered E. coli using untreated cane molasses. The recombinant E. coli cells expressing the SIase gene were immobilized in calcium alginate gel in order to improve the efficiency of recycling. The immobilization was most effective with 2 % (w/v) sodium alginate and 3 % (w/v) calcium chloride. The optimal initial biomass for immobilization was 20 % (w/v, wet wt.), with a hardening time of 8 h for cell immobilization. The immobilized E. coli cells exhibited good stability for 30 batches with the productivity of 0.45 g isomaltulose g pellet⁻¹ h⁻¹. A continuous isomaltulose formation process using a column reactor remained stable for 40 days with 83 ± 2 % isomaltulose yield, which would be beneficial for economical production of isomaltulose.

  13. Identifying inhibitory effects of lignocellulosic by-products on growth of lactic acid producing micro-organisms using a rapid small-scale screening method.

    PubMed

    van der Pol, Edwin C; Vaessen, Evelien; Weusthuis, Ruud A; Eggink, Gerrit

    2016-06-01

    Sugars obtained from pretreated lignocellulose are interesting as substrate for the production of lactic acid in fermentation processes. However, by-products formed during pretreatment of lignocellulose can inhibit microbial growth. In this study, a small-scale rapid screening method was used to identify inhibitory effects of single and combined by-products on growth of lactic acid producing micro-organisms. The small-scale screening was performed in 48-well plates using 5 bacterial species and 12 by-products. Large differences were observed in inhibitory effects of by-products between different species. Predictions can be made for growth behaviour of different micro-organisms on acid pretreated or alkaline pretreated bagasse substrates using data from the small-scale screening. Both individual and combined inhibition effects were shown to be important parameters to predict growth. Synergy between coumaric acid, formic acid and acetic acid is a key inhibitory parameter in alkaline pretreated lignocellulose, while furfural is a key inhibitor in acid pretreated lignocellulose. Copyright © 2016 Elsevier Ltd. All rights reserved.

  14. High Prevalence of Infectious Diseases and Drug-Resistant Microorganisms in Asylum Seekers Admitted to Hospital; No Carbapenemase Producing Enterobacteriaceae until September 2015

    PubMed Central

    Ravensbergen, Sofanne J.; Lokate, Mariëtte; Cornish, Darren; Kloeze, Eveline; Ott, Alewijn; Friedrich, Alex W.; van Hest, Rob; Akkerman, Onno W.; de Lange, Wiel C.; van der Werf, Tjip S.; Bathoorn, Erik

    2016-01-01

    Introduction The current refugee crisis emphasizes the need for information on infectious diseases and resistant microorganisms in asylum seekers with possible consequences for public health and infection control. Methods We collected data from asylum seekers admitted to our university hospital or who presented at the Emergency Department (n = 273). We collected general and demographic characteristics including country of origin, the reason of presentation, and the screening results of multi-drug resistant organisms. Results 67% of the patients were male with a median age of the study group of 24 years (IQR 15–33); 48% of the patients had an infectious disease—predominantly malaria with P. vivax or tuberculosis. Patients also reported with diseases which are less common—e.g. leishmaniasis, or even conditions rarely diagnosed in Europe—e.g. louse borne relapsing fever. A carriage rate of 31% for multi-drug resistant microorganisms (MDRO) was observed, with ESBL-expressing E.coli (n = 20) being the most common MDRO. No carriage of Carbapenemase Producing Enterobacteriaceae was found. Conclusion The current refugee crisis in Europe challenges hospitals to quickly identify and respond to communicable diseases and the carriage of MDRO. A rapid response is necessary to optimize the treatment of infectious diseases amongst asylum seekers to maximize infection control. PMID:27144599

  15. Transient Glyco-Engineering to Produce Recombinant IgA1 with Defined N- and O-Glycans in Plants

    PubMed Central

    Dicker, Martina; Tschofen, Marc; Maresch, Daniel; König, Julia; Juarez, Paloma; Orzaez, Diego; Altmann, Friedrich; Steinkellner, Herta; Strasser, Richard

    2016-01-01

    The production of therapeutic antibodies to combat pathogens and treat diseases, such as cancer is of great interest for the biotechnology industry. The recent development of plant-based expression systems has demonstrated that plants are well-suited for the production of recombinant monoclonal antibodies with defined glycosylation. Compared to immunoglobulin G (IgG), less effort has been undertaken to express immunoglobulin A (IgA), which is the most prevalent antibody class at mucosal sites and a promising candidate for novel recombinant biopharmaceuticals with enhanced anti-tumor activity. Here, we transiently expressed recombinant human IgA1 against the VP8* rotavirus antigen in glyco-engineered ΔXT/FT Nicotiana benthamiana plants. Mass spectrometric analysis of IgA1 glycopeptides revealed the presence of complex biantennary N-glycans with terminal N-acetylglucosamine present on the N-glycosylation site of the CH2 domain in the IgA1 alpha chain. Analysis of the peptide carrying nine potential O-glycosylation sites in the IgA1 alpha chain hinge region showed the presence of plant-specific modifications including hydroxyproline formation and the attachment of pentoses. By co-expression of enzymes required for initiation and elongation of human O-glycosylation it was possible to generate disialylated mucin-type core 1 O-glycans on plant-produced IgA1. Our data demonstrate that ΔXT/FT N. benthamiana plants can be engineered toward the production of recombinant IgA1 with defined human-type N- and O-linked glycans. PMID:26858738

  16. Transient Glyco-Engineering to Produce Recombinant IgA1 with Defined N- and O-Glycans in Plants.

    PubMed

    Dicker, Martina; Tschofen, Marc; Maresch, Daniel; König, Julia; Juarez, Paloma; Orzaez, Diego; Altmann, Friedrich; Steinkellner, Herta; Strasser, Richard

    2016-01-01

    The production of therapeutic antibodies to combat pathogens and treat diseases, such as cancer is of great interest for the biotechnology industry. The recent development of plant-based expression systems has demonstrated that plants are well-suited for the production of recombinant monoclonal antibodies with defined glycosylation. Compared to immunoglobulin G (IgG), less effort has been undertaken to express immunoglobulin A (IgA), which is the most prevalent antibody class at mucosal sites and a promising candidate for novel recombinant biopharmaceuticals with enhanced anti-tumor activity. Here, we transiently expressed recombinant human IgA1 against the VP8* rotavirus antigen in glyco-engineered ΔXT/FT Nicotiana benthamiana plants. Mass spectrometric analysis of IgA1 glycopeptides revealed the presence of complex biantennary N-glycans with terminal N-acetylglucosamine present on the N-glycosylation site of the CH2 domain in the IgA1 alpha chain. Analysis of the peptide carrying nine potential O-glycosylation sites in the IgA1 alpha chain hinge region showed the presence of plant-specific modifications including hydroxyproline formation and the attachment of pentoses. By co-expression of enzymes required for initiation and elongation of human O-glycosylation it was possible to generate disialylated mucin-type core 1 O-glycans on plant-produced IgA1. Our data demonstrate that ΔXT/FT N. benthamiana plants can be engineered toward the production of recombinant IgA1 with defined human-type N- and O-linked glycans.

  17. Prokaryotic High-Level Expression System in Producing Adhesin Recombinant Protein E of Nontypeable Haemophilus influenzae

    PubMed Central

    Tavakoli, Minoo; Bouzari, Saeed; Siadat, Seyed Davar; Najar Peerayeh, Shahin; Jafari, Anis

    2015-01-01

    Background: Adhesion protein E (PE) of Haemophilus influenzae is a 16 - 18 kDa protein with 160 amino acids which causes adhesion to epithelial cells and acts as a major factor in pathogenesis. Objectives: In this study, we performed cloning, expression and purification of PE as a candidate antigen for vaccine design upon further study. Materials and Methods: At first, the pe gene of NTHi ATCC 49766 strain (483 bp) was amplified by PCR. Then, to sequence the resulted amplicon, it was cloned into TA vector (pTZ57R/T). In the next step, the sequenced gene was sub-cloned in pBAD/gIII A vector and transformed into competent Escherichia coli TOP10. For overexpression, the recombinant bacteria were grown in broth medium containing arabinose and the recombinant protein was purified using metal affinity chromatography (Ni-nitrilotriacetic acid) (Ni-NTA agarose). Finally, the protein was detected using sodium dodecyl sulfate polyacrylamide gel electrophores (SDS-PAG) and confirmed by western blotting. Results: The cloned gene was confirmed by PCR, restriction digestion and sequencing. The sequenced gene was searched for homology in GenBank and 99% similarity was found to the already deposited genes in GenBank. Then we obtained PE using Ni-NTA agarose with up to 7 mg/mL concentration. Conclusions: The pe gene was successfully cloned and confirmed by sequencing. Finally, PE was obtained with high concentration. Due to high homology and similarity among the pe gene from NTHi ATCC 49766 and other NTHi strains in GenBank, we believe that the protein is a universal antigen to be used as a vaccine design candidate and further studies to evaluate its immunogenicity is underway. PMID:26034537

  18. Fat-free yogurt made using a galactose-positive exopolysaccharide-producing recombinant strain of Streptococcus thermophilus.

    PubMed

    Robitaille, G; Tremblay, A; Moineau, S; St-Gelais, D; Vadeboncoeur, C; Britten, M

    2009-02-01

    To prevent textural defects in low-fat and fat-free yogurts, fat substitutes are routinely added to milk. In situ production of exopolysaccharides (EPS) by starter cultures is an acknowledged alternative to the addition of biothickeners. With the aim of increasing in situ EPS production, a recombinant galactose-positive EPS(+) Streptococcus thermophilus strain, RD-534-S1, was generated and compared with the parent galactose-negative EPS(+) strain RD-534. The RD-534-S1 strain produced up to 84 mg/L of EPS during a single-strain milk fermentation process, which represented 1.3 times more than the EPS produced by strain RD-534. Under conditions that mimic industrial yogurt production, the starter culture consisting of RD-534-S1 and (EPS(-)) Lactobacillus bulgaricus L210R strain (RD-534-S1/L210R) led to an EPS production increase of 1.65-fold as compared with RD-534-S1 alone. However, the amount of EPS produced did not differ from that found in yogurts produced using an isogenic starter culture that included the parent S. thermophilus strain RD-534 and Lb. bulgaricus L210R (RD-534/L210R). Moreover, the gel characteristics of set-style yogurt and the rheological properties of stirred-style yogurt produced using RD-534-S1/L210R were similar to the values obtained for yogurts made with RD-534/L210R. In conclusion, it is possible to increase the production of EPS by ropy S. thermophilus strains through genetic engineering of galactose metabolism. However, when used in combination with Lb. bulgaricus for yogurt manufacture, the EPS overproduction of recombinant strain is not significant.

  19. [Pathways of degradation of organic components of waste water of (meth)acrylate-producing factories to methane by communities of microorganisms of adapted and unadapted sludge].

    PubMed

    Shtarkman, N B; Laurinavichius, K S

    1992-01-01

    Pathways of the degradation of the main compounds of (meth)acrylate-producing factories wastewater (methyl methacrylate, methyl and butyl acrylate, acrylate and methacrylate, acetone, isopropanol, butanol and methanol) by the anaerobic microbial consortium of mesophilic unadapted granulated sludge from the "UASB" reactor and of adapted activated sludge from the contact reactor were comparatively studied. It was shown that the degradation of fatty acids and alcohols took place in both types of sludge. Methacrylate, acrylate and acetone degradation occurred only in adapted sludge. Both types of sludge were characterized by the reversible conversion of acetone and isopropanol and by the presence of the isomeric transition of butyrate and isobutyrate too. The present results allow to suggest that the adaptation of activated sludge to substrate includes the accumulation of biomass of microorganisms capable of hydrolyze specific substrates into such general intermediates as low-molecular-weight fatty acid and alcohols further metabolized to methane and carbon dioxide.

  20. H2 and acetate transfers during xylan fermentation between a butyrate-producing xylanolytic species and hydrogenotrophic microorganisms from the human gut.

    PubMed

    Chassard, Christophe; Bernalier-Donadille, Annick

    2006-01-01

    The aim of this work was to investigate in vitro interrelationships during xylan fermentation between an H2 and butyrate-producing xylanolytic species recently isolated in our laboratory from human faeces and identified as Roseburia intestinalis and the H2-utilizing acetogen Ruminococcus hydrogenotrophicus or the methanogen Methanobrevibacter smithii. H2 transfer between M. smithii or Ru. hydrogenotrophicus and the xylanolytic species was evidenced, confirming the great potential of these H2-consuming microorganisms to reutilize fermentative H2 during fibre fermentation in the gut. In addition, acetate transfer was demonstrated between the xylanolytic Roseburia sp. and the acetogenic species, both metabolites transfers leading to butyric fermentation of oat xylan without production of H2.

  1. Dendritic Cell Activity Driven by Recombinant Mycobacterium bovis BCG Producing Human IL-18, in Healthy BCG Vaccinated Adults.

    PubMed

    Szpakowski, Piotr; Biet, Franck; Locht, Camille; Paszkiewicz, Małgorzata; Rudnicka, Wiesława; Druszczyńska, Magdalena; Allain, Fabrice; Fol, Marek; Pestel, Joël; Kowalewicz-Kulbat, Magdalena

    2015-01-01

    Tuberculosis remains an enormous global burden, despite wide vaccination coverage with the Bacillus Calmette-Guérin (BCG), the only vaccine available against this disease, indicating that BCG-driven immunity is insufficient to protect the human population against tuberculosis. In this study we constructed recombinant BCG producing human IL-18 (rBCGhIL-18) and investigated whether human IL-18 produced by rBCGhIL-18 modulates DC functions and enhances Th1 responses to mycobacterial antigens in humans. We found that the costimulatory CD86 and CD80 molecules were significantly upregulated on rBCGhIL-18-infected DCs, whereas the stimulation of DCs with nonrecombinant BCG was less effective. In contrast, both BCG strains decreased the DC-SIGN expression on human DCs. The rBCGhIL-18 increased IL-23, IL-10, and IP-10 production by DCs to a greater extent than nonrecombinant BCG. In a coculture system of CD4(+) T cells and loaded DCs, rBCGhIL-18 favoured strong IFN-γ but also IL-10 production by naive T cells but not by memory T cells. This was much less the case for nonrecombinant BCG. Thus the expression of IL-18 by recombinant BCG increases IL-23, IP-10, and IL-10 expression by human DCs and enhances their ability to induce IFN-γ and IL-10 expression by naive T cells, without affecting the maturation phenotype of the DCs.

  2. Dendritic Cell Activity Driven by Recombinant Mycobacterium bovis BCG Producing Human IL-18, in Healthy BCG Vaccinated Adults

    PubMed Central

    Biet, Franck; Rudnicka, Wiesława; Druszczyńska, Magdalena; Fol, Marek; Pestel, Joël

    2015-01-01

    Tuberculosis remains an enormous global burden, despite wide vaccination coverage with the Bacillus Calmette-Guérin (BCG), the only vaccine available against this disease, indicating that BCG-driven immunity is insufficient to protect the human population against tuberculosis. In this study we constructed recombinant BCG producing human IL-18 (rBCGhIL-18) and investigated whether human IL-18 produced by rBCGhIL-18 modulates DC functions and enhances Th1 responses to mycobacterial antigens in humans. We found that the costimulatory CD86 and CD80 molecules were significantly upregulated on rBCGhIL-18-infected DCs, whereas the stimulation of DCs with nonrecombinant BCG was less effective. In contrast, both BCG strains decreased the DC-SIGN expression on human DCs. The rBCGhIL-18 increased IL-23, IL-10, and IP-10 production by DCs to a greater extent than nonrecombinant BCG. In a coculture system of CD4+ T cells and loaded DCs, rBCGhIL-18 favoured strong IFN-γ but also IL-10 production by naive T cells but not by memory T cells. This was much less the case for nonrecombinant BCG. Thus the expression of IL-18 by recombinant BCG increases IL-23, IP-10, and IL-10 expression by human DCs and enhances their ability to induce IFN-γ and IL-10 expression by naive T cells, without affecting the maturation phenotype of the DCs. PMID:26339658

  3. Transglucosylation with 6'-chloro-6'-deoxysucrose and immobilized isomaltulose-producing microorganisms using 2,2-dimethyl-1,3-dioxolane-4-methanol and its related compounds as acceptors. Steric and chemical requirement of the glucosyl acceptor.

    PubMed

    Kakinuma, H; Tsuchiya, Y; Tanaka, M; Horito, S; Hashimoto, H

    1994-11-15

    Enantioselective and diastereoselective alpha-D-glucosylation of 2,3-O-isopropylidene-erythritol was observed in transglucosylation with a synthetic donor using three kinds of immobilized isomaltulose-producing microorganisms. Several related compounds, including an 2,3-O-isopropylidenated aldotetrose dimethyl dithioacetal and an aldotetronic acid ester were also glucosylated in moderate or good yield, depending on the microorganism utilized. Steric as well as functional group factors are discussed in relation to the substrate specificity of the glucosyl acceptor.

  4. HPLC quantification of biogenic amines in cheeses: correlation with PCR-detection of tyramine-producing microorganisms.

    PubMed

    Fernández, María; Linares, Daniel M; Del Río, Beatriz; Ladero, Victor; Alvarez, Miguel A

    2007-08-01

    The consumption of food and beverages containing high amounts of biogenic amines (BA) can have toxicological effects. BA found in foods and beverages are synthesized by the microbial decarboxylation of certain amino acids. This paper reports the concentrations of BAs in a number of commercial cheeses, as determined by HPLC. The cheeses studied were made from raw and pasteurized milk of different origin, and were subjected to different ripening periods. BA concentrations were lower in short ripening period than in long ripening period cheeses, and higher in cheeses made from raw milk than in those made from pasteurized milk. The highest BA concentrations were recorded in blue cheeses made from raw milk. Tyramine was the most commonly recorded and abundant BA. The presence of tyramine-producing bacteria was determined by PCR, and a good correlation obtained between the results of this method and tyramine detection by HPLC. These methods could be used to complement one another in the detection and quantification of tyramine in cheese prevention of tyramine accumulation in cheese.

  5. Hydrodynamic behavior of shaking flasks used for producing a recombinant protein by filamentous bacteria

    NASA Astrophysics Data System (ADS)

    Cordova Aguilar, Maria Soledad; Garcia, Monica; Trujillo-Roldan, Mauricio Alberto; Ascanio, Gabriel; Zenit, Roberto; Soto, Enrique

    2012-11-01

    Shake flasks are widely used for culture research. The agitation rate is one of the factors that determines the mass transfer. However, it has not been studied in detail. In this work, a comparison of the hydrodynamic performance for conventional, baffled and coiled spring Erlenmeyer flasks is presented. The velocity fields for a horizontal plane were measured by means of a Particle Image Velocimetry (PIV) technique and high speed videos were recorded to observe the behavior of the interface as a function of the agitation rate. It was observed not only that there is a strong dependence between the geometry and the hydrodynamics, but also there is a good agreement with the results obtained previously by Gamboa et al., in 2011, with the evaluation of the influence of culture conditions of S. lividans on protein O-glycosylation. The turbulence intensity increases with shaken rate. However, for the baffled geometry, it was observed a decrease for a critical speed, which is related with the in-phase and out-phase regions. These results can be an explanation for the variations in protein productivity as a function of the flask geometry and the differences in aggregation morphology and the pattern of O-glycosylation of the recombinant protein.

  6. Polysome profiling of mAb producing CHO cell lines links translational control of cell proliferation and recombinant mRNA loading onto ribosomes with global and recombinant protein synthesis.

    PubMed

    Godfrey, Charlotte L; Mead, Emma J; Daramola, Olalekan; Dunn, Sarah; Hatton, Diane; Field, Ray; Pettman, Gary; Smales, C Mark

    2017-08-01

    mRNA translation is a key process determining growth, proliferation and duration of a Chinese hamster ovary (CHO) cell culture and influences recombinant protein synthesis rate. During bioprocessing, CHO cells can experience stresses leading to reprogramming of translation and decreased global protein synthesis. Here we apply polysome profiling to determine reprogramming and translational capabilities in host and recombinant monoclonal antibody-producing (mAb) CHO cell lines during batch culture. Recombinant cell lines with the fastest cell specific growth rates were those with the highest global translational efficiency. However, total ribosomal capacity, determined from polysome profiles, did not relate to the fastest growing or highest producing mAb cell line, suggesting it is the ability to utilise available machinery that determines protein synthetic capacity. Cell lines with higher cell specific productivities tended to have elevated recombinant heavy chain transcript copy numbers, localised to the translationally active heavy polysomes. The highest titre cell line was that which sustained recombinant protein synthesis and maintained high recombinant transcript copy numbers in polysomes. Investigation of specific endogenous transcripts revealed a number that maintained or reprogrammed into heavy polysomes, identifying targets for potential cell engineering or those with 5' untranslated regions that might be utilised to enhance recombinant transcript translation. © 2017 The Authors. Biotechnology Journal published by WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  7. Plant cell calcium-rich environment enhances thermostability of recombinantly produced alpha-amylase from the hyperthermophilic bacterium Thermotoga maritime.

    PubMed

    Santa-Maria, Monica C; Chou, Chung-Jung; Yencho, G Craig; Haigler, Candace H; Thompson, William F; Kelly, Robert M; Sosinski, Bryon

    2009-12-01

    In the industrial processing of starch for sugar syrup and ethanol production, a liquefaction step is involved where starch is initially solubilized at high temperature and partially hydrolyzed with a thermostable and thermoactive alpha-amylase. Most amylases require calcium as a cofactor for their activity and stability, therefore calcium, along with the thermostable enzyme, are typically added to the starch mixture during enzymatic liquefaction, thereby increasing process costs. An attractive alternative would be to produce the enzyme directly in the tissue to be treated. In a proof of concept study, tobacco cell cultures were used as model system to test in planta production of a hyperthermophilic alpha-amylase from Thermotoga maritima. While comparable biochemical properties to recombinant production in Escherichia coli were observed, thermostability of the plant-produced alpha-amylase benefited significantly from high intrinsic calcium levels in the tobacco cells. The plant-made enzyme retained 85% of its initial activity after 3 h incubation at 100 degrees C, whereas the E. coli-produced enzyme was completely inactivated after 30 min under the same conditions. The addition of Ca(2+) or plant cell extracts from tobacco and sweetpotato to the E. coli-produced enzyme resulted in a similar stabilization, demonstrating the importance of a calcium-rich environment for thermostability, as well as the advantage of producing this enzyme directly in plant cells where calcium is readily available.

  8. Purification and physiochemical properties of a recombinant bovine growth hormone produced by cultured murine fibroblasts.

    PubMed

    Leung, F C; Jones, B; Steelman, S L; Rosenblum, C I; Kopchick, J J

    1986-10-01

    Mouse fibroblast cell lines which secrete bovine (b) GH have been generated. This was accomplished by cotransforming mouse L cells (thymidine kinase-negative [TK-] and adenine phosphoribosyl transferase-negative [APRT-]) with DNA molecules encoding the Rous sarcoma virus-long-terminal repeat and bGH genes along with the herpes viral TK gene and the hamster APRT gene. One stable cell line, Pd lambda-pbGH 4-13, was found to secrete approximately 75 micrograms bGH per 24 h/5.0 X 10(6) cells. Media from this cell line were collected for purification of recombinant bGH (rbGH). Purification involved (NH4)2SO4 fractionation, ion-exchange chromatography, and gel filtration on Sephacryl S-200. The rbGH was characterized by bioassay, RIA, radioreceptor assay, and sodium dodecyl sulfate gel electrophoresis. Results of these analyses were compared with those obtained with a highly purified pituitary bGH. In the rat tibia bioassay, rbGH was found to be as potent as pituitary bGH. Results from the RIA, radioreceptor assay, and sodium dodecyl sulfate gel electrophoresis and Western blot analysis also suggested that the rbGH was similar to that of pituitary origin. Amino acid composition, partial (amino-terminal) sequence, and tryptic peptide maps were also found to be similar between the rbGH and pituitary bGH preparations. The amino terminus of the rbGH showed similar heterogeneity to that of the bGH of pituitary origin. We conclude that rbGH which was synthesized, processed, and secreted from transformed mouse fibroblasts possessed almost exactly the same physiochemical properties as pituitary bGH.

  9. Aspergillus fumigatus Produces Two Arabinofuranosidases From Glycosyl Hydrolase Family 62: Comparative Properties of the Recombinant Enzymes.

    PubMed

    Pérez, Rodrigo; Eyzaguirre, Jaime

    2016-04-01

    The genes of two α-L-arabinofuranosidases (AbfI and II) from family GH 62 have been identified in the genome of Aspergillus fumigatus wmo. Both genes have been expressed in Pichia pastoris and the enzymes have been purified and characterized. AbfI is composed of 999 bp, does not contain introns and codes for a protein (ABFI) of 332 amino acid residues. abfII has 1246 bp, including an intron of 51 bp; the protein ABFII has 396 amino acid residues; it includes a family 1 carbohydrate-binding module (CBM) in the N-terminal region, followed by a catalytic module. The sequence of ABFI and the catalytic module of ABFII show a 79 % identity. Both enzymes are active on p-nitrophenyl α-L-arabinofuranoside (pNPAra) with KM of 94.2 and 3.9 mM for ABFI and II, respectively. Optimal temperature for ABFI is 37 °C and for ABFII 42 °C, while the pH optimum is about 4.5 to 5 for both enzymes. ABFII shows a higher thermostability. When assayed using natural substrates, both show higher activity over rye arabinoxylan as compared to wheat arabinoxylan. ABFII only is active on sugar beet pulp arabinan and both are inactive towards debranched arabinan. The higher thermostability, higher affinity for pNPAra and wider activity over natural substrates shown by ABFII may be related to the presence of a CBM. The availability of the recombinant enzymes may be useful in biotechnological applications for the production of arabinose.

  10. Partition separation and characterization of the polyhydroxyalkanoates synthase produced from recombinant Escherichia coli using an aqueous two-phase system.

    PubMed

    Lan, John Chi-Wei; Yeh, Chun-Yi; Wang, Chih-Chi; Yang, Yu-Hsuan; Wu, Ho-Shing

    2013-10-01

    Polyhydroxyalkanoates (PHAs) are renewable and biodegradable polyesters which can be synthesized either by numerous of microorganisms in vivo or synthase in vitro. The synthesis of PHAs in vitro requires an efficient separation for high yield of purified enzyme. The recombinant Escherichia coli harboring phaC gene derived from Ralstonia eutropha H16 was cultivated in the chemically defined medium for overexpression of synthase in the present work. The purification and characteristics of PHA synthase from clarified feedstock by using aqueous two-phase systems (ATPS) was investigated. The optimized concentration of ATPS for partitioning PHA synthase contained polyethylene glycol 6000 (30%, w/w) and potassium phosphate (8%, w/w) with 3.25 volume ratio in the absence of NaCl at pH 8.7 and 4°C. The results showed that the partition coefficient of enzyme activity and protein content are 6.07 and 0.22, respectively. The specific activity, selectivity, purification fold and recovery of phaC(Re) achieved 1.76 U mg⁻¹, 29.05, 16.23 and 95.32%, respectively. Several metal ions demonstrated a significant effect on activity of purified enzyme. The purified enzyme displayed maximum relative activity as operating condition at pH value of 7.5 and 37°C. As compared to conventional purification processes, ATPS can be a promising technique applied for rapid recovery of PHA synthase and preparation of large quantity of PHA synthase on synthesis of P(3HB) in vitro. Copyright © 2013 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.

  11. Sexual reproduction and recombination in the aflatoxin-producing fungus Aspergillus parasiticus

    USDA-ARS?s Scientific Manuscript database

    The fungal phylum Ascomycota comprises a large proportion of species with no known sexual stage, despite high genetic variability in field populations. One such asexual species, Aspergillus parasiticus, is a potent producer of carcinogenic and hepatotoxic aflatoxins, polyketide-derived secondary me...

  12. Plant-Produced Human Recombinant Erythropoietic Growth Factors Support Erythroid Differentiation In Vitro

    PubMed Central

    Musiychuk, Konstantin; Sivalenka, Rajarajeswari; Jaje, Jennifer; Bi, Hong; Flores, Rosemary; Shaw, Brenden; Jones, R. Mark; Golovina, Tatiana; Schnipper, Jacob; Khandker, Luipa; Sun, Ruiqiang; Li, Chang; Kang, Lin; Voskinarian-Berse, Vanessa; Zhang, Xiaokui; Streatfield, Stephen; Hambor, John; Abbot, Stewart

    2013-01-01

    Clinically available red blood cells (RBCs) for transfusions are at high demand, but in vitro generation of RBCs from hematopoietic stem cells requires significant quantities of growth factors. Here, we describe the production of four human growth factors: erythropoietin (EPO), stem cell factor (SCF), interleukin 3 (IL-3), and insulin-like growth factor-1 (IGF-1), either as non-fused proteins or as fusions with a carrier molecule (lichenase), in plants, using a Tobacco mosaic virus vector-based transient expression system. All growth factors were purified and their identity was confirmed by western blotting and peptide mapping. The potency of these plant-produced cytokines was assessed using TF1 cell (responsive to EPO, IL-3 and SCF) or MCF-7 cell (responsive to IGF-1) proliferation assays. The biological activity estimated here for the cytokines produced in plants was slightly lower or within the range cited in commercial sources and published literature. By comparing EC50 values of plant-produced cytokines with standards, we have demonstrated that all four plant-produced growth factors stimulated the expansion of umbilical cord blood-derived CD34+ cells and their differentiation toward erythropoietic precursors with the same potency as commercially available growth factors. To the best of our knowledge, this is the first report on the generation of all key bioactive cytokines required for the erythroid development in a cost-effective manner using a plant-based expression system. PMID:23517237

  13. Expression of recombinant organophosphorus hydrolase in the original producer of the enzyme, Sphingobium fuliginis ATCC 27551.

    PubMed

    Nakayama, Kosuke; Ohmori, Takeshi; Ishikawa, Satoshi; Iwata, Natsumi; Seto, Yasuo; Kawahara, Kazuyoshi

    2016-05-01

    The plasmid encoding His-tagged organophosphorus hydrolase (OPH) cloned from Sphingobium fuliginis was modified to be transferred back to this bacterium. The replication function of S. amiense plasmid was inserted at downstream of OPH gene, and S. fuliginis was transformed with this plasmid. The transformant produced larger amount of active OPH with His-tag than E. coli.

  14. A Recombinant Human Anti-Platelet scFv Antibody Produced in Pichia pastoris for Atheroma Targeting

    PubMed Central

    Vallet-Courbin, Amelie; Larivière, Mélusine; Hocquellet, Agnès; Hemadou, Audrey; Parimala, Sarjapura-Nagaraja; Laroche-Traineau, Jeanny; Santarelli, Xavier; Clofent-Sanchez, Gisèle; Jacobin-Valat, Marie-Josée; Noubhani, Abdelmajid

    2017-01-01

    Cells of the innate and adaptive immune system are key factors in the progression of atherosclerotic plaque, leading to plaque instability and rupture, potentially resulting in acute atherothrombotic events such as coronary artery disease, cerebrovascular disease and peripheral arterial disease. Here, we describe the cloning, expression, purification, and immunoreactivity assessment of a recombinant single-chain variable fragment (scFv) derived from a human anti-αIIbβ3 antibody (HuAb) selected to target atheromatous lesions for the presence of platelets. Indeed, platelets within atheroma plaques have been shown to play a role in inflammation, in platelet-leucocyte aggregates and in thrombi formation and might thus be considered relevant biomarkers of atherosclerotic progression. The DNA sequence that encodes the anti-αIIbβ3 TEG4 scFv previously obtained from a phage-display selection on activated platelets, was inserted into the eukaryote vector (pPICZαA) in fusion with a tag sequence encoding 2 cysteines useable for specific probes grafting experiments. The recombinant protein was expressed at high yields in Pichia pastoris (30 mg/L culture). The advantage of P. pastoris as an expression system is the production and secretion of recombinant proteins in the supernatant, ruling out the difficulties encountered when scFv are produced in the cytoplasm of bacteria (low yield, low solubility and reduced affinity). The improved conditions allowed for the recovery of highly purified and biologically active scFv fragments ready to be grafted in a site-directed way to nanoparticles for the imaging of atherosclerotic plaques involving inflammatory processes and thus at high risk of instability. PMID:28125612

  15. Real time detection of anthrax spores using highly specific anti-EA1 recombinant antibodies produced by competitive panning.

    PubMed

    Love, Tracey E; Redmond, Caroline; Mayers, Carl N

    2008-05-20

    We describe a targeted approach for the production of biological recognition elements capable of fast, specific detection of anthrax spores on biosensor surfaces. The aim was to produce single chain antibodies (scFvs) to EA1, a Bacillus anthracis S-layer protein that is also present, although not identical, in related to Bacillus species. The aim of the work was to produce antibodies that would detect B. anthracis EA1 protein and intact spores with a high degree of specificity, but would not detect other Bacillus species. Existing monoclonal antibodies were evaluated and found to recognise B. anthracis EA1 and S-layer proteins from other closely related Bacillus species. Recombinant anti-EA1 scFvs were isolated from B. anthracis immune library that contained antibody genes raised against B. anthracis spores and purified exosporium. Two approaches for scFv selection were used; standard (non-competitive) panning, and competitive panning. The non-competitive biopanning strategy isolated scFvs that recognised EA1 from B. anthracis, but also cross-reacted with other Bacillus species. In contrast, the competitive panning approach used S-layer proteins from other Bacillus species to generate scFvs that were highly specific to B. anthracis EA1 and demonstrated apparent nanomolar binding affinities. Specific, real time detection of B. anthracis spores was demonstrated with these scFvs using an evanescent wave biosensor, the Resonant Mirror. The approach described can be used to generate specific antibodies to any desired target where homologous proteins also exist in closely related species, and demonstrates clear advantages to using recombinant technology to produce biological recognition elements for detection of biological threat agents.

  16. The bifunctional enzyme chitosanase-cellulase produced by the gram-negative microorganism Myxobacter sp. AL-1 is highly similar to Bacillus subtilis endoglucanases.

    PubMed

    Pedraza-Reyes, M; Gutiérrez-Corona, F

    1997-10-01

    The gram-negative bacterium Myxobacter sp. AL-1 produces chitosanase-cellulase activity that is maximally excreted during the stationary phase of growth. Carboxymethylcellulase zymogram analysis revealed that the enzymatic activity was correlated with two bands of 32 and 35 kDa. Ion-exchange-chromatography-enriched preparations of the 32-kDa enzyme were capable of degrading the cellulose fluorescent derivatives 4-methylumbelliferyl-beta-D-cellobioside and 4-methylumbelliferyl-beta-D-cellotrioside. These enzymatic preparations also showed a greater capacity at 70 degrees C than at 42 degrees C to degrade chitosan oligomers of a minimum size of six units. Conversely, the beta-1,4 glucanolytic activity was more efficient at attacking carboxymethylcellulose and methylumbelliferyl-cellotrioside at 42 degrees C than at 70 degrees C. The 32-kDa enzyme was purified more than 800-fold to apparent homogeneity by a combination of ion-exchange and molecular-exclusion chromatography. Amino-terminal sequencing indicated that mature chitosanase-cellulase shares more than 70% identity with endocellulases produced by strains DLG, PAP115, and 168 of the gram-positive microorganism Bacillus subtilis.

  17. Optimizing promoters and secretory signal sequences for producing ethanol from inulin by recombinant Saccharomyces cerevisiae carrying Kluyveromyces marxianus inulinase.

    PubMed

    Hong, Soo-Jeong; Kim, Hyo Jin; Kim, Jin-Woo; Lee, Dae-Hee; Seo, Jin-Ho

    2015-02-01

    Inulin is a polyfructan that is abundant in plants such as Jerusalem artichoke, chicory and dahlia. Inulinase can easily hydrolyze inulin to fructose, which is consumed by microorganisms. Generally, Saccharomyces cerevisiae, an industrial workhorse strain for bioethanol production, is known for not having inulinase activity. The inulinase gene from Kluyveromyces marxianus (KmINU), with the ability of converting inulin to fructose, was introduced into S. cerevisiae D452-2. The inulinase gene was fused to three different types of promoter (GPD, PGK1, truncated HXT7) and secretory signal sequence (KmINU, MFα1, SUC2) to generate nine expression cassettes. The inulin fermentation performance of the nine transformants containing different promoter and signal sequence combinations for inulinase production were compared to select an optimized expression system for efficient inulin fermentation. Among the nine inulinase-producing transformants, the S. cerevisiae carrying the PGK1 promoter and MFα1 signal sequence (S. cerevisiae D452-2/p426PM) showed not only the highest specific KmINU activity, but also the best inulin fermentation capability. Finally, a batch fermentation of the selected S. cerevisiae D452-2/p426PM in a bioreactor with 188.2 g/L inulin was performed to produce 80.2 g/L ethanol with 0.43 g ethanol/g inulin of ethanol yield and 1.22 g/L h of ethanol productivity.

  18. Co-expression of ferrochelatase allows for complete heme incorporation into recombinant proteins produced in E. coli.

    PubMed

    Sudhamsu, Jawahar; Kabir, Mariam; Airola, Michael V; Patel, Bhumit A; Yeh, Syun-Ru; Rousseau, Denis L; Crane, Brian R

    2010-09-01

    Over-expression of heme binding proteins in Escherichia coli often results in sub-optimal heme incorporation and the amount of heme-bound protein produced usually varies with the protein of interest. Complete heme incorporation is important for biochemical characterization, spectroscopy, structural studies, and for the production of homogeneous commercial proteins with high activity. We have determined that recombinant proteins expressed in E. coli often contain less than a full complement of heme because they rather are partially incorporated with free-base porphyrin. Porphyrin-incorporated proteins have similar spectral characteristics as the desired heme-loaded targets, and thus are difficult to detect, even in purified samples. We present a straightforward and inexpensive solution to this problem that involves the co-expression of native ferrochelatase with the protein of interest. The method is shown to be effective for proteins that contain either Cys- or His-ligated hemes.

  19. A Cell Line Producing Recombinant Nerve Growth Factor Evokes Growth Responses in Intrinsic and Grafted Central Cholinergic Neurons

    NASA Astrophysics Data System (ADS)

    Ernfors, Patrik; Ebendal, Ted; Olson, Lars; Mouton, Peter; Stromberg, Ingrid; Persson, Hakan

    1989-06-01

    The rat β nerve growth factor (NGF) gene was inserted into a mammalian expression vector and cotransfected with a plasmid conferring resistance to neomycin into mouse 3T3 fibroblasts. From this transfection a stable cell line was selected that contains several hundred copies of the rat NGF gene and produces excess levels of recombinant NGF. Such genetically modified cells were implanted into the rat brain as a probe for in vivo effects of NGF on central nervous system neurons. In a model of the cortical cholinergic deficits in Alzheimer disease, we demonstrate a marked increase in the survival of, and fiber outgrowth from, grafts of fetal basal forebrain cholinergic neurons, as well as stimulation of fiber formation by intact adult intrinsic cholinergic circuits in the cerebral cortex. Adult cholinergic interneurons in intact striatum also sprout vigorously toward implanted fibroblasts. Our results suggest that this model has implications for future treatment of neurodegenerative diseases.

  20. Glutamic acid decarboxylase autoantibody assay using 125I-labelled recombinant GAD65 produced in yeast.

    PubMed

    Powell, M; Prentice, L; Asawa, T; Kato, R; Sawicka, J; Tanaka, H; Petersen, V; Munkley, A; Morgan, S; Rees Smith, B; Furmaniak, J

    1996-12-30

    We describe a new method for measuring autoantibodies (Ab) to the 65 kDa isoform of glutamic acid carboxylase (GAD65). In particular, GAD65 without the hydrophobic N-terminal region has been produced in yeast, purified, labelled with 125I and reacted with GAD65 Ab. Antibody bound 125I-GAD65 is then precipitated by the addition of solid phase protein A. With the assay, GAD65 Ab were detected in 59 of 71 (83%) islet cell antibody (ICA) positive IDDM patients and in 8 of 23 (35%) ICA negative IDDM patients (overall 67 of 94 (71%) of IDDM patients). Low concentrations of GAD65 Ab were also detected in 2/98 (2%) healthy blood donors and 1/27 (4%) Graves' disease patients had a high level of antibody. GAD65 Ab were not detected in any of 10 Hashimoto's thyroiditis, 20 Addison's disease or 19 myasthenia gravis sera. There was good agreement between the 125I assay and the current reference method based on 35S-labelled full-length GAD65 (produced by in vitro transcription/translation reaction) and solid phase protein A (r = 0.91, n = 108). Overall, our 125I assay showed sensitivity, precision and disease group specificity at least as good as any assay so far described. These features, combined with a simple assay protocol and the convenience of 125I counting and handling indicate that the method is suitable for routine GAD65 Ab measurements.

  1. Recombinant anthrax toxin receptor-Fc fusion proteins produced in plants protect rabbits against inhalational anthrax.

    PubMed

    Wycoff, Keith L; Belle, Archana; Deppe, Dorothée; Schaefer, Leah; Maclean, James M; Haase, Simone; Trilling, Anke K; Liu, Shihui; Leppla, Stephen H; Geren, Isin N; Pawlik, Jennifer; Peterson, Johnny W

    2011-01-01

    Inhalational anthrax, a zoonotic disease caused by the inhalation of Bacillus anthracis spores, has a ∼50% fatality rate even when treated with antibiotics. Pathogenesis is dependent on the activity of two toxic noncovalent complexes: edema toxin (EdTx) and lethal toxin (LeTx). Protective antigen (PA), an essential component of both complexes, binds with high affinity to the major receptor mediating the lethality of anthrax toxin in vivo, capillary morphogenesis protein 2 (CMG2). Certain antibodies against PA have been shown to protect against anthrax in vivo. As an alternative to anti-PA antibodies, we produced a fusion of the extracellular domain of human CMG2 and human IgG Fc, using both transient and stable tobacco plant expression systems. Optimized expression led to the CMG2-Fc fusion protein being produced at high levels: 730 mg/kg fresh leaf weight in Nicotiana benthamiana and 65 mg/kg in N. tabacum. CMG2-Fc, purified from tobacco plants, fully protected rabbits against a lethal challenge with B. anthracis spores at a dose of 2 mg/kg body weight administered at the time of challenge. Treatment with CMG2-Fc did not interfere with the development of the animals' own immunity to anthrax, as treated animals that survived an initial challenge also survived a rechallenge 30 days later. The glycosylation of the Fc (or lack thereof) had no significant effect on the protective potency of CMG2-Fc in rabbits or on its serum half-life, which was about 5 days. Significantly, CMG2-Fc effectively neutralized, in vitro, LeTx-containing mutant forms of PA that were not neutralized by anti-PA monoclonal antibodies.

  2. Specificity of antibodies produced against native or desialylated human immunodeficiency virus type 1 recombinant gp160.

    PubMed Central

    Benjouad, A; Gluckman, J C; Montagnier, L; Bahraoui, E

    1993-01-01

    In a previous report we have shown that, in contrast to antibodies produced against native or fully deglycosylated human immunodeficiency virus type 1 (HIV-1) gp160 in rabbits, antibodies raised against desialylated HIV-1 gp160 also recognize gp140 from HIV-2 at high titers. Here, we characterize the fine specificity of these cross-reactive antibodies. Inhibition assays with a panel of synthetic peptides as competitors showed that cross-reactivity to gp140 was due to antibodies that were specific for the region encompassing HIV-1 gp41 immunodominant epitope, mimicked by peptide P39 (residues 583 to 609), the latter being able to totally inhibit the formation of complexes between radiolabeled HIV-2 gp140 and antibodies elicited by desialylated HIV-1 gp160. In addition, anti-desialylated gp160 antibodies retained on a P39 affinity column still bound HIV-2 gp140. Fine mapping has enabled us to localize the cross-reactive epitope within the N-terminal extremity of the gp41 immunodominant region. Interestingly, this cross-reactive antibody population did not recognize glycosylated or totally deglycosylated simian immunodeficiency virus gp140 despite an amino acid homology with HIV-1 within this region that is comparable to that of HIV-2. This cross-reactivity between HIV-1 and HIV-2 did not correlate with cross-neutralization. These results illustrate the influence of carbohydrate moieties on the specificity of the antibodies produced and clearly indicate that such procedures may be an efficient way to raise specific immune responses that are not type specific. Moreover, this cross-reactivity might explain the double-positive reactivity observed, in some human sera, against both HIV-1 and HIV-2 envelope antigens. PMID:7679751

  3. A Recombinant Fungal Chitin Deacetylase Produces Fully Defined Chitosan Oligomers with Novel Patterns of Acetylation.

    PubMed

    Naqvi, Shoa; Cord-Landwehr, Stefan; Singh, Ratna; Bernard, Frank; Kolkenbrock, Stephan; El Gueddari, Nour Eddine; Moerschbacher, Bruno M

    2016-11-15

    Partially acetylated chitosan oligosaccharides (paCOS) are potent biologics with many potential applications, and their bioactivities are believed to be dependent on their structure, i.e., their degrees of polymerization and acetylation, as well as their pattern of acetylation. However, paCOS generated via chemical N-acetylation or de-N-acetylation of GlcN or GlcNAc oligomers, respectively, typically display random patterns of acetylation, making it difficult to control and predict their bioactivities. In contrast, paCOS produced from chitin deacetylases (CDAs) acting on chitin oligomer substrates may have specific patterns of acetylation, as shown for some bacterial CDAs. However, compared to what we know about bacterial CDAs, we know little about the ability of fungal CDAs to produce defined paCOS with known patterns of acetylation. Therefore, we optimized the expression of a chitin deacetylase from the fungus Puccinia graminis f. sp. tritici in Escherichia coli The best yield of functional enzyme was obtained as a fusion protein with the maltose-binding protein (MBP) secreted into the periplasmic space of the bacterial host. We characterized the MBP fusion protein from P. graminis (PgtCDA) and tested its activity on different chitinous substrates. Mass spectrometric sequencing of the products obtained by enzymatic deacetylation of chitin oligomers, i.e., tetramers to hexamers, revealed that PgtCDA generated paCOS with specific acetylation patterns of A-A-D-D, A-A-D-D-D, and A-A-D-D-D-D, respectively (A, GlcNAc; D, GlcN), indicating that PgtCDA cannot deacetylate the two GlcNAc units closest to the oligomer's nonreducing end. This unique property of PgtCDA significantly expands the so far very limited library of well-defined paCOS available to test their bioactivities for a wide variety of potential applications.

  4. Effects of recombinant methionyl bovine somatotropin (sometribove) in high producing cows milked three times daily.

    PubMed

    Jordan, D C; Aguilar, A A; Olson, J D; Bailey, C; Hartnell, G F; Madsen, K S

    1991-01-01

    Effects of daily sometribove administration on milk yield and composition, body condition score, BW, and SCC were evaluated in Holstein cows milked three times daily. Lactating cows (n =104) were assigned randomly to control or sometribove-treated (25 mg/d) groups. The experimental period was 16 wk, consisting of 2-wk pretreatment, 12-wk treatment, and 2-wk posttreatment periods. All cows were injected once daily starting at 53 to 180 d postpartum, housed in free stalls, and fed one of five total mixed rations according to milk production. Body weights were measured weekly, and body condition was scored biweekly. Milk yield was recorded daily, and weekly milk samples were analyzed for fat, protein, lactose, total solids, and SCC. Milk yield and milk protein were increased 18.8% (38.6 vs. 32.5 kg/d) and 3.3% (3.1 vs. 3.0%), respectively, whereas percentage of milk fat, lactose, SNF, SCC, and BW were unaffected by treatment. Overall average body condition scores were lower for the sometribove-treated group versus control (2.2 vs. 2.4). No apparent differences in the number of cows treated for mastitis, foot rot, displaced abomasum, or lameness were observed between treatment groups. Sometribove treatment significantly enhanced milk yield (6.1 kg/d) with no apparent negative effects on health in high producing cows milked three times per day.

  5. Quantitative Determination of Bandpasses for Producing Vegetation Indices from Recombined NEON Hyperspectral Imagery

    NASA Astrophysics Data System (ADS)

    Hulslander, D.

    2015-12-01

    Hyperspectral imaging systems can be used to produce spectral reflectance curves giving rich information about composition, relative abundances of materials, mixes and combinations. However, as each spectral return from these systems is a vector with several hundred elements, they can be very difficult to process and analyze, and problemeatic to compare within, across, and between datasets over time and space. Vegetation indices (e.g. NDVI, ARVI, EVI, et al) attempt to combine spectral features in to single-value scores. When derived from calibrated and atmospherically compensated reflectance data, these indices can be quantitatively compared. Historically, these indices have been calculated from multispectral sensor data. These sensors have a handful (4 to 16 or so) of bandbasses ranging from 20 nm to 200 nm FWHM covering specific spectral regions for a variety of reasons, including both intended applications and system limitations. Hyperspectral sensors, however, cover the spectrum with many, many narrow (5 to 10 nm) bandpasses. This allows for analyses using the full, detailed spectral curve, or combination of the bands in to regions by averaging or in to composites using transforms or other techniques. This raises the question of exactly which bands should be used and combined in what manner for ideally deriving well-known vegetation indices typically made from multispectral data. In this study we use derivatives and other curve and signal analysis techniques to analyze vegetation reflectance spectra to quantitatively define optimal bandpasses for several vegetation indices and combine the 5 nm hypserspectral bandpasses of the NEON Imaging Spectrometer to synthesize them.

  6. Validation of the manufacturing process used to produce long-acting recombinant factor IX Fc fusion protein.

    PubMed

    McCue, J; Osborne, D; Dumont, J; Peters, R; Mei, B; Pierce, G F; Kobayashi, K; Euwart, D

    2014-07-01

    Recombinant factor IX Fc (rFIXFc) fusion protein is the first of a new class of bioengineered long-acting factors approved for the treatment and prevention of bleeding episodes in haemophilia B. The aim of this work was to describe the manufacturing process for rFIXFc, to assess product quality and to evaluate the capacity of the process to remove impurities and viruses. This manufacturing process utilized a transferable and scalable platform approach established for therapeutic antibody manufacturing and adapted for production of the rFIXFc molecule. rFIXFc was produced using a process free of human- and animal-derived raw materials and a host cell line derived from human embryonic kidney (HEK) 293H cells. The process employed multi-step purification and viral clearance processing, including use of a protein A affinity capture chromatography step, which binds to the Fc portion of the rFIXFc molecule with high affinity and specificity, and a 15 nm pore size virus removal nanofilter. Process validation studies were performed to evaluate identity, purity, activity and safety. The manufacturing process produced rFIXFc with consistent product quality and high purity. Impurity clearance validation studies demonstrated robust and reproducible removal of process-related impurities and adventitious viruses. The rFIXFc manufacturing process produces a highly pure product, free of non-human glycan structures. Validation studies demonstrate that this product is produced with consistent quality and purity. In addition, the scalability and transferability of this process are key attributes to ensure consistent and continuous supply of rFIXFc. © 2014 The Authors. Haemophilia Published by John Wiley & Sons Ltd.

  7. Validation of the manufacturing process used to produce long-acting recombinant factor IX Fc fusion protein

    PubMed Central

    McCue, J; Osborne, D; Dumont, J; Peters, R; Mei, B; Pierce, G F; Kobayashi, K; Euwart, D

    2014-01-01

    Recombinant factor IX Fc (rFIXFc) fusion protein is the first of a new class of bioengineered long-acting factors approved for the treatment and prevention of bleeding episodes in haemophilia B. The aim of this work was to describe the manufacturing process for rFIXFc, to assess product quality and to evaluate the capacity of the process to remove impurities and viruses. This manufacturing process utilized a transferable and scalable platform approach established for therapeutic antibody manufacturing and adapted for production of the rFIXFc molecule. rFIXFc was produced using a process free of human- and animal-derived raw materials and a host cell line derived from human embryonic kidney (HEK) 293H cells. The process employed multi-step purification and viral clearance processing, including use of a protein A affinity capture chromatography step, which binds to the Fc portion of the rFIXFc molecule with high affinity and specificity, and a 15 nm pore size virus removal nanofilter. Process validation studies were performed to evaluate identity, purity, activity and safety. The manufacturing process produced rFIXFc with consistent product quality and high purity. Impurity clearance validation studies demonstrated robust and reproducible removal of process-related impurities and adventitious viruses. The rFIXFc manufacturing process produces a highly pure product, free of non-human glycan structures. Validation studies demonstrate that this product is produced with consistent quality and purity. In addition, the scalability and transferability of this process are key attributes to ensure consistent and continuous supply of rFIXFc. PMID:24811361

  8. Novel Feruloyl Esterase from Lactobacillus fermentum NRRL B-1932 and Analysis of the Recombinant Enzyme Produced in Escherichia coli

    PubMed Central

    Bischoff, Kenneth M.; Anderson, Amber M.; Rich, Joseph O.

    2016-01-01

    ABSTRACT A total of 33 Lactobacillus strains were screened for feruloyl esterase (FE) activity using agar plates containing ethyl ferulate as the sole carbon source, and Lactobacillus fermentum NRRL B-1932 demonstrated the strongest FE activity among a dozen species showing a clearing zone on the opaque plate containing ethyl ferulate. FE activities were monitored using high-performance liquid chromatography with an acetonitrile-trifluoroacetic acid gradient. To produce sufficient purified FE from L. fermentum strain NRRL B-1932 (LfFE), the cDNA encoding LfFE (Lffae) was amplified and cloned by using available closely related genome sequences and overexpressed in Escherichia coli. A 29.6-kDa LfFE protein was detected from the protein extract of E. coli BL21(pLysS) carrying pET28bLffae upon IPTG (isopropyl-β-d-thiogalactopyranoside) induction. The recombinant LfFE containing a polyhistidine tag was purified by nickel-nitrilotriacetic acid affinity resin. The purified LfFE showed strong activities against several artificial substrates, including p-nitrophenyl acetate and 4-methylumbelliferyl p-trimethylammoniocinnamate chloride. The optimum pH and temperature of the recombinant LfFE were around 6.5 and 37°C, respectively, as determined using either crude or purified recombinant LfFE. This study will be essential for the production of the LfFE in E. coli on a larger scale that could not be readily achieved by L. fermentum fermentation. IMPORTANCE The production of feruloyl esterase (FE) from Lactobacillus fermentum NRRL B-1932 reported in this study will have immense potential commercial applications not only in biofuel production but also in pharmaceutical, polymer, oleo chemical, cosmetic additive, and detergent industries, as well as human health-related applications, including food flavoring, functional foods, probiotic agents, preventive medicine, and animal feed. Given the essential role FE plays in the production of hydroxycinnamic acids and ferulic acid

  9. [Producing recombinant adenovirus encoding green fluorescent protein (Ad-GFP) by suspension cultured HEK-293 N3S cells].

    PubMed

    Tian, Bo; Wu, Bin; Zhang, Qun-Wei; Bi, Jian-Jin; Wang, Lan; Zhu, Bao-Zhen; Geng, Yue; Wu, Zu-Ze

    2007-09-01

    Adenovirus vectors are one of the most promising gene transfer systems. They are of great value for gene therapy because these vectors achieve temporal high-level transgene expression and high gene transfer efficiency. To meet increasing needs of adenovirus vectors for gene therapy programs, parallel development of efficient, scalable and reproducible production processes is required. Perfusion cultivation of 293 cells is one of the most commonly used methods to produce adenovirus vectors and it is suitable for industrialized production specially. Experimental studies had been carried out to produce recombinant adenovirus containing the green fluorescent protein gene (Ad-GFP) by perfusion cultivation of HEK-293 N3S cells in a 5L stirring bioreactors. Perfusion rate was 1-2 volume/day. To infect the 293 N3S cells with Ad-GFP at the density of (2-4) x 10(6) cells/ ml. The time of collecting cells was 48 hours post infection. After three rounds of freeze/thaw and centrifugation, the crude viral lysates were stored at--80 degrees C until use. Then to get the Ad-GFP products by 2 x CsCl-gradient purification. The purity of the products was determined by the A260/A280 ratio and a high performance liquid chromatography (HPLC) assay. The infective titer was determined by a TCID50 assay. The culture term was 10-12 days. The infectious titer, the number of virus particle and the ratio of infectious titer to virus particle for the product were 1.0 x 10(11) IU/mL, 1.68 x 10(12) VP/mL and 6.0% IU/VP respectively. The A260/A280 ratio was 1.33, and the purity determined by HPLC was 99.2%. The cell specific productivity was around 1000 IU/cell. By perfusion cultivation of 293 N3S cells in a 5L stirring bioreactors, we established the production process for Ad-GFP, which paves a way to produce other recombinant adenovirus for gene therapy.

  10. Causes of proteolytic degradation of secreted recombinant proteins produced in methylotrophic yeast Pichia pastoris: case study with recombinant ovine interferon-tau.

    PubMed

    Sinha, Jayanta; Plantz, Bradley A; Inan, Mehmet; Meagher, Michael M

    2005-01-05

    It was observed that during fermentative production of recombinant ovine interferon-tau (r-oIFN-tau) in Pichia pastoris, a secreted recombinant protein, the protein was degraded increasingly after 48 h of induction and the rate of degradation increased towards the end of fermentation at 72 h, when the fermentation was stopped. Proteases, whose primary source was the vacuoles, was found in increasing levels in the cytoplasm and in the fermentation broth after 48 h of induction and reached maximal values when the batch was completed at 72 h. Protease levels at various cell fractions as well as in the culture supernatant were lower when glycerol was used as the carbon source instead of methanol. It can be concluded that methanol metabolism along with cell lysis towards the end of fermentation contributes to increased proteolytic activity and eventual degradation of recombinant protein.

  11. Effects of amino acid on morphological development and nucleus formation of arachidonic acid-producing filamentous micro-organism, Mortierella alpina.

    PubMed

    Koizumi, K; Higashiyama, K; Park, E Y

    2006-04-01

    Effects of amino acid on morphological development and nucleus formation of arachidonic acid-producing filamentous micro-organism, Mortierella alpina were investigated using flow-through chamber. Mortierella alpina CBS 754.68 was cultivated in flow through chamber using nutrient-rich, minimal and specific amino acid-containing minimal media. To investigate the effect of amino acid on morphological parameters either 0.28 g l(-1) alanine, 0.53 g l(-1) sodium glutamate one hydrate or 0.42 g l(-1) valine was added to the minimal medium. In a flow-through chamber, the growth of hyphal elements and nucleus formation of arachidonic acid-producing fungus M. alpina were studied on-line, using image analysis techniques. When the Ala- and Val-containing media were used, the hyphal growth units (HGUs) were 90.2 and 86.7 microm per tip, respectively, which were 2.4-fold higher than that in the nutrient-rich medium, indicating that Ala and Val stimulate the elongation of hyphae. The specific nucleus formation rates were Glu->Val-containing media>minimal and nutrient-rich media>Ala-containing medium. The nucleus doubling times in Glu- and Val-containing media were 1.9 and 2 h, respectively, which were not significant different. Ala and Val stimulate the elongation of M. alpina hyphae, and nucleus formation rates were Glu->Val->Ala-containing media. Formation of fungal morphology and nucleus were shown using the flow-through chamber coupled with image analysis, which making possible to discuss the relationship between mycelial morphology and nucleus formation of M. alpina.

  12. Escherichia coli can produce recombinant chitinase in the soil to control the pathogenesis by Fusarium oxysporum without colonization.

    PubMed

    Chung, Soohee; Kim, Sang-Dal

    2007-03-01

    Fusarium wilt of cucumbers was effectively controlled by Escherichia coli expressing an endochitinase gene (chiA), and the rate was as effective (60.0%) as the wildtype strain S. proteamaculans 3095 (55.0%) where the gene was cloned. However, live cells of soil inoculated E. coli host harboring the chiA gene did not proliferate but declined 100-fold from 108 CFU during the first week and showed less than 10 cells after day 14, suggesting that E. coli was able to express and produce the chitinase enzyme to the soil even as the population was gradually decreasing. Because the majority of the strains was alive for only a short period of time and the Fusarium-affected seedlings showed symptoms of wilting within 7-10 days, it seems that the pathogen control was decided early after the introduction of the biocontrol agent, eliminating the survival of the antagonist. These results indicated that soil inoculated E. coli could sufficiently express and produce the recombinant protein to control the pathogen, and root or soil colonization of the antagonist might not be a significant factor in determining the efficacy of biological control.

  13. Effect of recombinant Lactococcus lactis producing myelin peptides on neuroimmunological changes in rats with experimental allergic encephalomyelitis.

    PubMed

    Kasarełło, K; Szczepankowska, A; Kwiatkowska-Patzer, B; Lipkowski, A W; Gadamski, R; Sulejczak, D; Łachwa, M; Biały, M; Bardowski, J

    2016-01-01

    Multiple sclerosis (MS) is a human autoimmune neurodegenerative disease with an unknown etiology. Despite various therapies, there is no effective cure for MS. Since the mechanism of the disease is based on autoreactive T-cell responses directed against myelin antigens, oral tolerance is a promising approach for the MS treatment. Here, the experiments were performed to assess the impact of oral administration of recombinant Lactococcus lactis producing encephalogenic fragments of three myelin proteins: myelin basic protein, proteolipid protein, and myelin oligodendrocyte glycoprotein, on neuroimmunological changes in rats with experimental allergic encephalomyelitis (EAE) - an animal model of MS. Lactococcus lactis whole-cell lysates were administered intragastrically at two doses (103 and 106 colony forming units) in a twenty-fold feeding regimen to Lewis rats with EAE. Spinal cord slices were subjected to histopathological analysis and morphometric evaluation, and serum levels of cytokines (IL-1b, IL-10, TNF-α and IFN-γ) were measured. Results showed that administration of the L. lactis preparations at the tested doses to rats with EAE, diminished the histopathological changes observed in EAE rats and reduced the levels of serum IL-1b, IL-10 and TNF-α, previously increased by evoking EAE. This suggests that oral delivery of L. lactis producing myelin peptide fragments could be an alternative strategy to induce oral tolerance for the treatment of MS.

  14. High-level expression and purification of recombinant human growth hormone produced in soluble form in Escherichia coli.

    PubMed

    Levarski, Zdenko; Šoltýsová, Andrea; Krahulec, Ján; Stuchlík, Stanislav; Turňa, Ján

    2014-08-01

    Human growth hormone (hGH) was one of the first recombinant proteins approved for the treatment of human growth disorders. Its small size (191 amino acids), possession of only 2 disulphide bonds and absence of posttranslational modifications make Escherichia coli the host of choice for its production on any scale. In this work, we have utilized an efficient T7 based expression system to produce high levels of soluble thioredoxin-hGH (Trx-hGH) fusion protein. We outline a relatively simple three step purification process employing two immobilized metal-affinity chromatography and one anion-exchange steps and removal of fusion partner by enterokinase cleavage yielding native hGH. The ability of cell populations to produce quantities of up to 1 g/L of the soluble Trx-hGH fusion protein has been tested in flask cultivations as well as in batch and fed-batch bioreactor runs. The sequence and structure of derived hGH were confirmed by mass spectrometry and circular dichroism and its native function, to induce cell proliferation, was confirmed by employing a Nb2 cell line proliferation assay.

  15. Rapid, scalable, and low-cost purification of recombinant adeno-associated virus produced by baculovirus expression vector system

    PubMed Central

    Buclez, Pierre-Olivier; Dias Florencio, Gabriella; Relizani, Karima; Beley, Cyriaque; Garcia, Luis; Benchaouir, Rachid

    2016-01-01

    Recombinant adeno-associated viruses (rAAV) are largely used for gene transfer in research, preclinical developments, and clinical trials. Their broad in vivo biodistribution and long-term efficacy in postmitotic tissues make them good candidates for numerous gene transfer applications. Upstream processes able to produce large amounts of rAAV were developed, particularly those using baculovirus expression vector system. In parallel, downstream processes present a large panel of purification methods, often including multiple and time consuming steps. Here, we show that simple tangential flow filtration, coupled with an optimized iodixanol-based isopycnic density gradient, is sufficient to purify several liters of crude lysate produced by baculovirus expression vector system in only one working day, leading to high titers and good purity of rAAV products. Moreover, we show that the viral vectors retain their in vitro and in vivo functionalities. Our results demonstrate that simple, rapid, and relatively low-cost methods can easily be implemented for obtaining a high-quality grade of gene therapy products based on rAAV technology. PMID:27226971

  16. [Requirements to a medical and biologic assessment and the hygienic control of the food production received from recombinant-DNA microorganisms].

    PubMed

    Sheveleva, S A; Efimmochkina, N R; Nesterenko, L N; Zigangirova, N A; Khovaev, A A; Naroditskiĭ, B S; Ivanov, G E; Tutel'ian, V A; Gintsburg, A L

    2008-01-01

    In work the characteristic of the created in the Russian Federation system of an estimation of safety of the foodstuff received from/or with use of genetically modified microorganisms (GMM) is given, at their admission to realization and the hygienic control of given production over a revolution. It is shown, that strategy of a safety at a stage of registration GMM, the established order and accepted control measures of the foodstuff received from/or with use GMM, in Russia their large-scale commercial use, and the normative-legal and methodical base based on the federal legislation on state regulation in the field of genetically engineering activity, about quality and effectively outstrip safety of foodstuff about protection of the rights of consumers, is harmonized with approaches of the international organizations.

  17. Enzyme replacement therapy for Morquio A: an active recombinant N-acetylgalactosamine-6-sulfate sulfatase produced in Escherichia coli BL21.

    PubMed

    Rodríguez, Alexander; Espejo, Angela J; Hernández, Alejandra; Velásquez, Olga L; Lizaraso, Lina M; Cordoba, Henry A; Sánchez, Oscar F; Alméciga-Díaz, Carlos J; Barrera, Luis A

    2010-11-01

    Mucopolysaccharidosis IVA (MPS IVA) is an autosomal recessive disorder caused by N-acetylgalactosamine-6-sulfate sulfatase (GALNS) deficiency. Currently no effective therapies exist for MPS IVA. In this work, production of a recombinant GALNS enzyme (rGALNS) in Escherichia coli BL21 strain was studied. At shake scale, the effect of glucose concentration on microorganism growth, and microorganism culture and induction times on rGALNS production were evaluated. At bench scale, the effect of aeration and agitation on microorganism growth, and culture and induction times were evaluated. The highest enzyme activity levels at shake scale were observed in 12 h culture after 2-4 h induction. At bench scale the highest enzyme activity levels were observed after 2 h induction. rGALNS amounts in inclusion bodies fraction were up to 17-fold higher than those observed in the soluble fraction. However, the highest levels of active enzyme were found in the soluble fraction. Western blot analysis showed the presence of a 50-kDa band, in both soluble and inclusion bodies fractions. These results show for the first time the feasibility and potential of production of active rGALNS in a prokaryotic system for development of enzyme replacement therapy for MPS IVA disease.

  18. Medical devices; general and plastic surgery devices; classification of absorbable poly(hydroxybutyrate) surgical suture produced by recombinant DNA technology. Final rule.

    PubMed

    2007-08-03

    The Food and Drug Administration (FDA) is classifying the absorbable poly(hydroxybutyrate) surgical suture produced by recombinant deoxyribonucleic acid (DNA) technology into class II (special controls). The special control that will apply to the device is the guidance document entitled "Class II Special Controls Guidance Document: Absorbable Poly(hydroxybutyrate) Surgical Suture Produced by Recombinant DNA Technology." The agency is classifying these devices into class II (special controls) in order to provide a reasonable assurance of safety and effectiveness of these devices. Elsewhere in this issue of the Federal Register, FDA is announcing the availability of the guidance document that will serve as the special control for this device.

  19. Recombinant Nox4 cytosolic domain produced by a cell or cell-free base systems exhibits constitutive diaphorase activity

    SciTech Connect

    Nguyen, Minh Vu Chuong; Zhang, Leilei; Lhomme, Stanislas; Mouz, Nicolas

    2012-03-16

    Highlights: Black-Right-Pointing-Pointer A comparison of two bacterial cell and cell-free protein expression systems is presented. Black-Right-Pointing-Pointer Soluble and active truncated Nox4 proteins are produced. Black-Right-Pointing-Pointer Nox4 has a constitutive diaphorase activity which is independent of cytosolic factors. Black-Right-Pointing-Pointer Isoform Nox4B is unable to initiate the first electronic transfer step. Black-Right-Pointing-Pointer Findings contribute to the understanding of the mechanism of Nox4 oxidase activity. -- Abstract: The membrane protein NADPH (nicotinamide adenine dinucleotide phosphate) oxidase Nox4 constitutively generates reactive oxygen species differing from other NADPH oxidases activity, particularly in Nox2 which needs a stimulus to be active. Although the precise mechanism of production of reactive oxygen species by Nox2 is well characterized, the electronic transfer throughout Nox4 remains unclear. Our study aims to investigate the initial electronic transfer step (diaphorase activity) of the cytosolic tail of Nox4. For this purpose, we developed two different approaches to produce soluble and active truncated Nox4 proteins. We synthesized soluble recombinant proteins either by in vitro translation or by bacteria induction. While proteins obtained by bacteria induction demonstrate an activity of 4.4 {+-} 1.7 nmol/min/nmol when measured against iodonitro tetrazolium chloride and 20.5 {+-} 2.8 nmol/min/nmol with cytochrome c, the soluble proteins produced by cell-free expression system exhibit a diaphorase activity with a turn-over of 26 {+-} 2.6 nmol/min/nmol when measured against iodonitro tetrazolium chloride and 48 {+-} 20.2 nmol/min/nmol with cytochrome c. Furthermore, the activity of the soluble proteins is constitutive and does not need any stimulus. We also show that the cytosolic tail of the isoform Nox4B lacking the first NADPH binding site is unable to demonstrate any diaphorase activity pointing out the

  20. Preparation of Specific Polyclonal Antibody Against the Recombinant Mutacin Produced by sfGFP Fusion Protein Technology

    PubMed Central

    Al-Homsi, Lamis; Al-Okla, Souad; Abbady, Abdul Q.

    2015-01-01

    Mutacin I, a bacteriocin produced by streptococcus mutans, displays an antimicrobial activity against many gram positive and some gram negative bacteria. Because of its medical importance, production of this short peptide in large scale for future applications is a significant challenge. This work described the improvement of a novel system to produce the recombinant mutacin using fusion protein technology. The short peptide was expressed directly as a fusion protein with a superfolder form of the green florescent protein (sfGFP), resulting in a high yield expression of soluble sfGFP-mutacin fusion protein (30 kDa) in the cytoplasm of E. coli. Mutacin was released from the fusion by enzymatic cleavage at the tobacco etch virus (TEV) protease recognition site and separated from the carrier sfGFP by nickel affinity and gel filtration chromatography. An additional advantage of this fusion system was tested in the generation of mutacin-specific polyclonal antibodies. Specific anti-mutacin IgGs were affinity purified, and were able to recognize the mutacin-sfGFP fusion protein or the cleaved forms of mutacin. Even though it was efficiently produced (25 mg/L) by this method, pure mutacin was devoid of antibiotic activity. Fourier transform infrared spectroscopy (FTIR) analysis revealed the absence of thioether bonds in the purified mutacin, which are critical for final structure and function of this antibiotic. Determining whether the activity of pure mutacin could be recovered by the reformation of such structures by chemical reaction needs more investigations. The development of this system will provide large quantities of mutacin for future studies and applications as broad spectrum antibacterial peptide. PMID:26668664

  1. Biochemical characterization of LR769, a new recombinant factor VIIa bypassing agent produced in the milk of transgenic rabbits.

    PubMed

    Chevreux, G; Tilly, N; Leblanc, Y; Ramon, C; Faid, V; Martin, M; Dhainaut, F; Bihoreau, N

    2017-07-01

    The bypassing agent factor VII (FVIIa) is a first-line therapy for the treatment of acute bleeding episodes in patients with haemophilia and high-titre inhibitors. FVIIa is a highly post-translationally modified protein that requires eukaryotic expression systems to produce a fully active molecule. A recombinant FVIIa was produced in the milk of transgenic rabbits to increase expression and provide an efficient, safe and affordable product after purification to homogeneity (LR769). To present the biochemical and functional in vitro characteristics of LR769. Mass spectrometric analyses of the intact protein and of heavy and light chains revealed a fully activated, mature and properly post-translationally modified protein notably regarding N/O-glycosylations and γ-carboxylation. Primary structure analysis, performed by peptide mapping, confirmed 100% of the sequence and the low level or absence of product-derived impurities such as oxidized, deamidated and glycated forms. Low levels of aggregates and fragments were observed by different chromatographic methods. Higher order structure investigated by circular dichroism showed appropriate secondary/tertiary structures and conformational change in the presence of Ca(2+) ions. Finally, activated partial thromboplastin time and thrombin generation assays showed the ability of LR769 to decrease coagulation time and to generate thrombin in haemophiliac-A-plasmas, even in the presence of inhibitors. The innovative expression system used to produce LR769 yields a new safe and effective rhFVIIa for the treatment of haemophilia A or B patients with inhibitors. © 2017 LFB Biotechnologies. Haemophilia Published by John Wiley & Sons Ltd.

  2. Microorganism identification technique

    SciTech Connect

    Sillman, R. E.

    1985-07-02

    An identification technique for micro-organisms in which a dilute solution of a culture medium containing an unknown micro-organism has added thereto an emissive agent such as a radioactive amino acid to produce a mix of emissive products that depends on the metabolic mechanism of the micro-organism. After a predetermined incubation period, the reaction is arrested and the solution layered onto a gel plate where it is subjected to electrophoresis. The plate is then autoradiographed by exposing the gel to a sensitive photographic film for a period sufficient to produce thereon a characteristic band pattern functioning as an identifier for the micro-organism. Identification may be effected by comparing the identifier for the unknown with a collection of identifiers for known micro-organisms to find a match with one of these known identifiers. The comparison is preferably carried out by scanning the unknown identifier to produce a signal which is compared with signals representing known identifiers stored in a computer which, when a match is found, yields identification data. Alternatively, the emissive products, after separation, may be detected by direct scanning to provide an identifier signal for computer processing.

  3. Detailed analysis of metagenome datasets obtained from biogas-producing microbial communities residing in biogas reactors does not indicate the presence of putative pathogenic microorganisms

    PubMed Central

    2013-01-01

    Background In recent years biogas plants in Germany have been supposed to be involved in amplification and dissemination of pathogenic bacteria causing severe infections in humans and animals. In particular, biogas plants are discussed to contribute to the spreading of Escherichia coli infections in humans or chronic botulism in cattle caused by Clostridium botulinum. Metagenome datasets of microbial communities from an agricultural biogas plant as well as from anaerobic lab-scale digesters operating at different temperatures and conditions were analyzed for the presence of putative pathogenic bacteria and virulence determinants by various bioinformatic approaches. Results All datasets featured a low abundance of reads that were taxonomically assigned to the genus Escherichia or further selected genera comprising pathogenic species. Higher numbers of reads were taxonomically assigned to the genus Clostridium. However, only very few sequences were predicted to originate from pathogenic clostridial species. Moreover, mapping of metagenome reads to complete genome sequences of selected pathogenic bacteria revealed that not the pathogenic species itself, but only species that are more or less related to pathogenic ones are present in the fermentation samples analyzed. Likewise, known virulence determinants could hardly be detected. Only a marginal number of reads showed similarity to sequences described in the Microbial Virulence Database MvirDB such as those encoding protein toxins, virulence proteins or antibiotic resistance determinants. Conclusions Findings of this first study of metagenomic sequence reads of biogas producing microbial communities suggest that the risk of dissemination of pathogenic bacteria by application of digestates from biogas fermentations as fertilizers is low, because obtained results do not indicate the presence of putative pathogenic microorganisms in the samples analyzed. PMID:23557021

  4. Classifying Microorganisms.

    ERIC Educational Resources Information Center

    Baker, William P.; Leyva, Kathryn J.; Lang, Michael; Goodmanis, Ben

    2002-01-01

    Focuses on an activity in which students sample air at school and generate ideas about how to classify the microorganisms they observe. The results are used to compare air quality among schools via the Internet. Supports the development of scientific inquiry and technology skills. (DDR)

  5. Classifying Microorganisms.

    ERIC Educational Resources Information Center

    Baker, William P.; Leyva, Kathryn J.; Lang, Michael; Goodmanis, Ben

    2002-01-01

    Focuses on an activity in which students sample air at school and generate ideas about how to classify the microorganisms they observe. The results are used to compare air quality among schools via the Internet. Supports the development of scientific inquiry and technology skills. (DDR)

  6. Recombinant bovine dihydrofolate reductase produced by mutagenesis and nested PCR of murine dihydrofolate reductase cDNA.

    PubMed

    Cody, Vivian; Mao, Qilong; Queener, Sherry F

    2008-11-01

    Recent reports of the slow-tight binding inhibition of bovine liver dihydrofolate reductase (bDHFR) in the presence of polyphenols isolated from green tea leaves has spurred renewed interest in the biochemical properties of bDHFR. Earlier studies were done with native bDHFR but in order to validate models of polyphenol binding to bDHFR, larger quantities of bDHFR are necessary to support structural studies. Bovine DHFR differs from its closest sequence homologue, murine DHFR, by 19 amino acids. To obtain the bDHFR cDNA, murineDHFR cDNA was transformed by a series of nested PCRs to reproduce the amino acid coding sequence for bovine DHFR. The bovine liver DHFR cDNA has an open reading frame of 561 base pairs encoding a protein of 187 amino acids that has a high level of conservation at the primary sequence level with other DHFR enzymes, and more so for the amino acid residues in the active site of the mammalian DHFR enzymes. Expression of the bovine DHFR cDNA in bacterial cells produced a stable recombinant protein with high enzymatic activity and kinetic properties similar to those previously reported for the native protein.

  7. Growth of recombinant Drosophila melanogaster Schneider 2 cells producing rabies virus glycoprotein in bioreactor employing serum-free medium

    PubMed Central

    Galesi, Adriana L. L.; Aguiar, Marcelo A.; Astray, Renato M.; Augusto, Elisabeth F. P.

    2008-01-01

    Drosophila melanogaster Schneider 2 (S2) cells have been increasingly used as a suitable expression system for the production of different recombinant proteins, and the employment of bioreactors for large-scale culture is an important tool for this purpose. In this work, Drosophila S2 cells producing the rabies virus glycoprotein RVGP were cultivated in bioreactor, employing a serum-free medium, aiming an improvement in cell growth and in glycoprotein production. To overcome cell growth limitation commonly observed in stirred flasks, different experiments in bioreactor were performed, in which some system modifications were carried out to attain the desired goal. The study showed that this cell line is considerably sensitive to hydrodynamic forces, and a high cell density (about 16.0 × 106 cells mL−1) was only obtained when Pluronic F68® percentage was increased to 0.6% (w/v). Despite ammonium concentration affected RVGP production, and also cell growth, an elevated amount of the target protein was obtained, attaining 563 ng 10−7 cells. PMID:19003175

  8. Control of specific carbon dioxide production in a fed-batch culture producing recombinant protein using a soft sensor.

    PubMed

    Gustavsson, Robert; Lukasser, Cornelia; Mandenius, Carl-Fredrik

    2015-04-20

    The feeding of a fed-batch cultivation producing recombinant protein was controlled by a soft sensor set-up. It was assumed that the control approach could be based on the cell's production of carbon dioxide and that this parameter indicates the metabolic state occurring at induced protein expression. The soft sensor used the on-line signals from a carbon dioxide analyser and a near-infrared (NIR) probe for biomass to estimate the specific production rate qCO2. Control experiments were carried out with various qCO2 set-points where we observe that the feeding of nutrients to the culture could easily be controlled and resulted in a decreased variability compared to uncontrolled cultivations. We therefore suggest that this control approach could serve as an alternative to other commonly applied methods such as controlling the cell's overflow metabolism of acetate or the cell's specific growth rate. However, further studies of the internal metabolic fluxes of CO2 during protein expression would be recommended for a more precise characterization of the relationship between qCO2 and protein expression in order to fully interpret the control behaviour.

  9. In vivo efficacy of human recombinant factor IX produced by the human hepatoma cell line HuH-7.

    PubMed

    Enjolras, N; Perot, E; Le Quellec, S; Indalecio, A; Girard, J; Negrier, C; Dargaud, Y

    2015-07-01

    Post-translational modifications of the CHO-cell-derived-recombinant human factor IX (FIX) currently used for the treatment of hemophilia B (HB) are different from plasma derived FIX. Our previous studies described a rFIX (HIX) having better profile of post-translational modifications than rFIX produced by CHO cells. The aim of the study consisted to verify the improved post-translational modifications effect of HIX on in vivo recovery. HIX has been produced in a bioreactor and then purified from supernatants. In vitro activation and activity were evaluated measured by thrombin generation tests (TGT) and compared to commercial molecules, Benefix(®) , Mononine(®) . The three molecules were then administrated (i.v.) to FIX-knockout mice and two minutes after injection, blood samples were collected and subjected to human FIX-specific-ELISA and TGT. The clotting function of HIX, activation courses of HIX by FXIa and FVIIa-TF complex appear normal as did activation of Benefix(®) , Mononine(®) and TG constants of each FIX were equivalent. After injection to HB mice, circulating HIX did not present any significant difference in term of antigen value with Benefix(®) . Intriguingly, TGT were clearly exhibiting a better velocity for HIX than Benefix(®) and Mononine(®) . These data suggested that HIX may improve in vivo coagulant efficacy in comparison with the two commercial FIX injected at the same dose. The study shows that HuH-7-derived-rFIX has better in vivo haemostatic activity in hemophilia B mice compared to the reference rFIX molecule despite similar in vivo recovery rates, suggesting that HuH-7 cells could represent an effective cellular system for production of rFIX. © 2015 John Wiley & Sons Ltd.

  10. Recombinant Mycobacterium bovis BCG producing IL-18 reduces IL-5 production and bronchoalveolar eosinophilia induced by an allergic reaction.

    PubMed

    Biet, F; Duez, C; Kremer, L; Marquillies, P; Amniai, L; Tonnel, A-B; Locht, C; Pestel, J

    2005-08-01

    Allergic reactions occur through the exacerbated induction of a Th2 cell type expression profile and can be prevented by agents favoring a Th1 profile. Bacillus Calmette-Guérin (BCG) is able to induce high IFN-gamma levels and has been shown to decrease experimentally induced allergy. The induction of IFN-gamma is mediated by interleukin (IL)-12 known to be secreted upon mycobacterial infections and can be enhanced by IL-18 acting in synergy with IL-12. We evaluated the ability of a recombinant BCG strain producing IL-18 (rBCG) to modify the Th2 type responses in a murine model of ovalbumin (OVA)-dependent allergic reaction. Mice were injected intraperitoneally or intranasally with OVA at days 0 and 15 and exposed to an OVA aerosol challenge at days 29, 30, 31 and 34. At days 0 and 15, two additional groups of mice received OVA together with 5 x 10(6) colony forming units of either rBCG or nonrecombinant BCG. A time-course analysis of OVA-specific immunoglobulin (Ig)E, IgG1 and IgG2a levels indicated no significant difference between the three groups of mice. However, following in vitro stimulation with OVA, lymph node cells from rBCG-treated mice produced less IL-5 and more IFN-gamma than those of mice injected with nonrecombinant BCG. In addition, 48 h after the last OVA challenge, a strong reduction of bronchoalveolar eosinophilia was found in the rBCG-injected mice compared to the nontreated or nonrecombinant BCG-treated groups. These results indicate that the production of IL-18 by rBCG may enhance the immunomodulatory properties of BCG that suppress pulmonary Th2 responses and, in particular, decrease airway eosinophilia.

  11. Techno-economic analysis of a conceptual biofuel production process from bioethylene produced by photosynthetic recombinant cyanobacteria

    DOE PAGES

    Markham, Jennifer N.; Tao, Ling; Davis, Ryan; ...

    2016-08-25

    Ethylene is a petrochemical produced in large volumes worldwide. It serves as a building block for a wide variety of plastics, textiles, and chemicals, and can be converted into liquid transportation fuels. There is great interest in the development of technologies that produce ethylene from renewable resources, such as biologically derived CO2 and biomass. One of the metabolic pathways used by microbes to produce ethylene is via an ethylene-forming enzyme (EFE). By expressing a bacterial EFE gene in a cyanobacterium, ethylene has been produced through photosynthetic carbon fixation. Here, we present a conceptual design and techno-economic analysis of a processmore » of biofuel production based on the upgradation of ethylene generated by the recombinant cyanobacterium. This analysis focuses on potential near-term to long-term cost projections for the integrated process of renewable fuels derived from ethylene. The cost projections are important in showing the potential of this technology and determining research thrusts needed to reach target goals. The base case for this analysis is a midterm projection using tubular photobioreactors for cyanobacterial growth and ethylene production, cryogenic distillation for ethylene separation and purification, a two-step Ziegler oligomerization process with subsequent hydrotreatment and upgradation for fuel production, and a wastewater treatment process that utilizes anaerobic digestion of cyanobacterial biomass. The minimum fuel selling price (MFSP) for the midterm projection is 15.07 per gallon gasoline equivalent (GGE). Near-term and long-term projections are 28.66 per GGE and 5.36 per GGE, respectively. Single- and multi-point sensitivity analyses are conducted to determine the relative effect that chosen variables could have on the overall costs. This analysis identifies several key variables for improving the overall process economics and outlines strategies to guide future research directions. Finally, the

  12. Techno-economic analysis of a conceptual biofuel production process from bioethylene produced by photosynthetic recombinant cyanobacteria

    SciTech Connect

    Markham, Jennifer N.; Tao, Ling; Davis, Ryan; Voulis, Nina; Angenent, Largus T.; Ungerer, Justin; Yu, Jianping

    2016-08-25

    Ethylene is a petrochemical produced in large volumes worldwide. It serves as a building block for a wide variety of plastics, textiles, and chemicals, and can be converted into liquid transportation fuels. There is great interest in the development of technologies that produce ethylene from renewable resources, such as biologically derived CO2 and biomass. One of the metabolic pathways used by microbes to produce ethylene is via an ethylene-forming enzyme (EFE). By expressing a bacterial EFE gene in a cyanobacterium, ethylene has been produced through photosynthetic carbon fixation. Here, we present a conceptual design and techno-economic analysis of a process of biofuel production based on the upgradation of ethylene generated by the recombinant cyanobacterium. This analysis focuses on potential near-term to long-term cost projections for the integrated process of renewable fuels derived from ethylene. The cost projections are important in showing the potential of this technology and determining research thrusts needed to reach target goals. The base case for this analysis is a midterm projection using tubular photobioreactors for cyanobacterial growth and ethylene production, cryogenic distillation for ethylene separation and purification, a two-step Ziegler oligomerization process with subsequent hydrotreatment and upgradation for fuel production, and a wastewater treatment process that utilizes anaerobic digestion of cyanobacterial biomass. The minimum fuel selling price (MFSP) for the midterm projection is 15.07 per gallon gasoline equivalent (GGE). Near-term and long-term projections are 28.66 per GGE and 5.36 per GGE, respectively. Single- and multi-point sensitivity analyses are conducted to determine the relative effect that chosen variables could have on the overall costs. This analysis identifies several key variables for improving the overall process economics and outlines strategies to guide future research directions. Finally, the

  13. Techno-economic analysis of a conceptual biofuel production process from bioethylene produced by photosynthetic recombinant cyanobacteria

    SciTech Connect

    Markham, Jennifer N.; Tao, Ling; Davis, Ryan; Voulis, Nina; Angenent, Largus T.; Ungerer, Justin; Yu, Jianping

    2016-08-25

    Ethylene is a petrochemical produced in large volumes worldwide. It serves as a building block for a wide variety of plastics, textiles, and chemicals, and can be converted into liquid transportation fuels. There is great interest in the development of technologies that produce ethylene from renewable resources, such as biologically derived CO2 and biomass. One of the metabolic pathways used by microbes to produce ethylene is via an ethylene-forming enzyme (EFE). By expressing a bacterial EFE gene in a cyanobacterium, ethylene has been produced through photosynthetic carbon fixation. Here, we present a conceptual design and techno-economic analysis of a process of biofuel production based on the upgradation of ethylene generated by the recombinant cyanobacterium. This analysis focuses on potential near-term to long-term cost projections for the integrated process of renewable fuels derived from ethylene. The cost projections are important in showing the potential of this technology and determining research thrusts needed to reach target goals. The base case for this analysis is a midterm projection using tubular photobioreactors for cyanobacterial growth and ethylene production, cryogenic distillation for ethylene separation and purification, a two-step Ziegler oligomerization process with subsequent hydrotreatment and upgradation for fuel production, and a wastewater treatment process that utilizes anaerobic digestion of cyanobacterial biomass. The minimum fuel selling price (MFSP) for the midterm projection is 15.07 per gallon gasoline equivalent (GGE). Near-term and long-term projections are 28.66 per GGE and 5.36 per GGE, respectively. Single- and multi-point sensitivity analyses are conducted to determine the relative effect that chosen variables could have on the overall costs. This analysis identifies several key variables for improving the overall process economics and outlines strategies to guide future research directions. Finally, the

  14. Purification and characterization of Streptococcus sobrinus dextranase produced in recombinant Escherichia coli and sequence analysis of the dextranase gene.

    PubMed Central

    Wanda, S Y; Curtiss, R

    1994-01-01

    The plasmid (pYA902) with the dextranase (dex) gene of Streptococcus sobrinus UAB66 (serotype g) produces a C-terminal truncated dextranase enzyme (Dex) with a multicomplex mass form which ranges from 80 to 130 kDa. The Escherichia coli-produced enzyme was purified and characterized, and antibodies were raised in rabbits. Purified dextranase has a native-form molecular mass of 160 to 260 kDa and specific activity of 4,000 U/mg of protein. Potential immunological cross-reactivity between dextranase and the SpaA protein specified by various recombinant clones was studied by using various antisera and Western blot (immunoblot) analysis. No cross-reactivity was observed. Optimal pH (5.3) and temperature (39 degrees C) and the isoelectric points (3.56, 3.6, and 3.7) were determined and found to be similar to those for dextranase purified from S. sobrinus. The dex DNA restriction map was determined, and several subclones were obtained. The nucleotide sequence of the dex gene was determined by using subclones pYA993 and pYA3009 and UAB66 chromosomal DNA. The open reading frame for dex was 4,011 bp, ending with a stop codon TAA. A ribosome-binding site and putative promoter preceding the start codon were identified. The deduced amino acid sequence of Dex revealed the presence of a signal peptide of 30 amino acids. The cleavage site for the signal sequence was determined by N-terminal amino acid sequence analysis for Dex produced in E. coli chi 2831(pYA902). The C terminus consists of a serine- and threonine-rich region followed by the peptide LPKTGD, 3 charged amino acids, 19 amino acids with a strongly hydrophobic character, and a charged pentapeptide tail, which are proposed to correspond to the cell wall-spanning region, the LPXTGX consensus sequence, and the membrane-anchoring domains of surface-associated proteins of gram-positive cocci. Images PMID:8021165

  15. Anti-loxoscelic horse serum produced against a recombinant dermonecrotic protein of Brazilian Loxosceles intermedia spider neutralize lethal effects of Loxosceles laeta venom from Peru.

    PubMed

    Duarte, C G; Bonilla, C; Guimarães, G; Machado de Avila, R A; Mendes, T M; Silva, W; Tintaya, B; Yarleque, A; Chávez-Olórtegui, C

    2015-01-01

    In this work, an anti-loxoscelic serum was produced by immunizing horses with a recombinant dermonecrotic protein from Loxosceles intermedia (rLiD1). Anti-rLiD1 antibodies were able to recognize different species of Loxosceles venoms by Western Blot and ELISA. The efficacy of anti-rLiD1 serum against the toxic effects of Loxosceles laeta (Peru) venom was tested, showing that anti-rLiD1 serum can neutralize those effects. This study confirms that recombinant proteins can be good candidates to replace crude venoms for antivenom production. Copyright © 2014 Elsevier Ltd. All rights reserved.

  16. Industrial Microorganisms.

    ERIC Educational Resources Information Center

    Phaff, Herman J.

    1981-01-01

    Describes industrially important yeasts, molds, bacteria, and actinomycetes. Discussed in detail are microbial products, such as primary metabolites, secondary metabolites, enzymes, and capsular polysaccharides. Traces the historical background of human cell culture, mentioning recombinant DNA research and hybridization of normal mammalian cells…

  17. Industrial Microorganisms.

    ERIC Educational Resources Information Center

    Phaff, Herman J.

    1981-01-01

    Describes industrially important yeasts, molds, bacteria, and actinomycetes. Discussed in detail are microbial products, such as primary metabolites, secondary metabolites, enzymes, and capsular polysaccharides. Traces the historical background of human cell culture, mentioning recombinant DNA research and hybridization of normal mammalian cells…

  18. Scaled-up manufacturing of recombinant antibodies produced by plant cells in a 200-L orbitally-shaken disposable bioreactor.

    PubMed

    Raven, Nicole; Rasche, Stefan; Kuehn, Christoph; Anderlei, Tibor; Klöckner, Wolf; Schuster, Flora; Henquet, Maurice; Bosch, Dirk; Büchs, Jochen; Fischer, Rainer; Schillberg, Stefan

    2015-02-01

    Tobacco BY-2 cells have emerged as a promising platform for the manufacture of biopharmaceutical proteins, offering efficient protein secretion, favourable growth characteristics and cultivation in containment under a controlled environment. The cultivation of BY-2 cells in disposable bioreactors is a useful alternative to conventional stainless steel stirred-tank reactors, and orbitally-shaken bioreactors could provide further advantages such as simple bag geometry, scalability and predictable process settings. We carried out a scale-up study, using a 200-L orbitally-shaken bioreactor holding disposable bags, and BY-2 cells producing the human monoclonal antibody M12. We found that cell growth and recombinant protein accumulation were comparable to standard shake flask cultivation, despite a 200-fold difference in cultivation volume. Final cell fresh weights of 300-387 g/L and M12 yields of ∼20 mg/L were achieved with both cultivation methods. Furthermore, we established an efficient downstream process for the recovery of M12 from the culture broth. The viscous spent medium prevented clarification using filtration devices, but we used expanded bed adsorption (EBA) chromatography with SP Sepharose as an alternative for the efficient capture of the M12 antibody. EBA was introduced as an initial purification step prior to protein A affinity chromatography, resulting in an overall M12 recovery of 75-85% and a purity of >95%. Our results demonstrate the suitability of orbitally-shaken bioreactors for the scaled-up cultivation of plant cell suspension cultures and provide a strategy for the efficient purification of antibodies from the BY-2 culture medium.

  19. TALEN-Mediated Homologous Recombination Produces Site-Directed DNA Base Change and Herbicide-Resistant Rice.

    PubMed

    Li, Ting; Liu, Bo; Chen, Chih Ying; Yang, Bing

    2016-05-20

    Over the last decades, much endeavor has been made to advance genome editing technology due to its promising role in both basic and synthetic biology. The breakthrough has been made in recent years with the advent of sequence-specific endonucleases, especially zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs) and clustered regularly interspaced short palindromic repeats (CRISPRs) guided nucleases (e.g., Cas9). In higher eukaryotic organisms, site-directed mutagenesis usually can be achieved through non-homologous end-joining (NHEJ) repair to the DNA double-strand breaks (DSBs) caused by the exogenously applied nucleases. However, site-specific gene replacement or genuine genome editing through homologous recombination (HR) repair to DSBs remains a challenge. As a proof of concept gene replacement through TALEN-based HR in rice (Oryza sativa), we successfully produced double point mutations in rice acetolactate synthase gene (OsALS) and generated herbicide resistant rice lines by using TALENs and donor DNA carrying the desired mutations. After ballistic delivery into rice calli of TALEN construct and donor DNA, nine HR events with different genotypes of OsALS were obtained in T0 generation at the efficiency of 1.4%-6.3% from three experiments. The HR-mediated gene edits were heritable to the progeny of T1 generation. The edited T1 plants were as morphologically normal as the control plants while displayed strong herbicide resistance. The results demonstrate the feasibility of TALEN-mediated genome editing in rice and provide useful information for further genome editing by other nuclease-based genome editing platforms.

  20. Recombinant human IGF-1 produced by transgenic plant cell suspension culture enhances new bone formation in calvarial defects.

    PubMed

    Poudel, Sher Bahadur; Bhattarai, Govinda; Kook, Sung-Ho; Shin, Yun-Ji; Kwon, Tae-Ho; Lee, Seung-Youp; Lee, Jeong-Chae

    2017-07-30

    Transgenic plant cell suspension culture systems have been utilized extensively as convenient and efficient expression systems for the production of recombinant human growth factors. We produced insulin-like growth factor-1 using a plant suspension culture system (p-IGF-1) and explored its effect on new bone formation in calvarial defects. We also compared the bone regenerating potential of p-IGF-1 with commercial IGF-1 derived from Escherichia coli (e-IGF-1). Male C57BL/6 mice underwent calvarial defect surgery, and the defects were loaded with absorbable collagen sponge (ACS) only (ACS group) or ACS impregnated with 13μg of p-IGF-1 (p-IGF-1 group) or e-IGF-1 (e-IGF-1 group). The sham group did not receive any treatment with ACS or IGFs after surgery. Live μCT and histological analyses showed critical-sized bone defects in the sham group, whereas greater bone formation was observed in the p-IGF-1 and e-IGF-1 groups than the ACS group both 5 and 10weeks after surgery. Bone mineral density, bone volume, and bone surface values were also higher in the IGF groups than in the ACS group. Local delivery of p-IGF-1 or e-IGF-1 more greatly enhanced the expression of osteoblast-specific markers, but inhibited osteoclast formation, in newly formed bone compared with ACS control group. Specifically, p-IGF-1 treatment induced higher expression of alkaline phosphatase, osteocalcin, and osteopontin in the defect site than did e-IGF-1. Furthermore, treatment with p-IGF-1, but not e-IGF-1, increased mineralization of MC3T3-E1 cells, with the attendant upregulation of osteogenic marker genes. Collectively, our findings suggest the potential of p-IGF-1 in promoting the processes required for bone regeneration. Copyright © 2017. Published by Elsevier Ltd.

  1. Development of microorganisms for cellulose-biofuel consolidated bioprocessings: metabolic engineers’ tricks

    PubMed Central

    Mazzoli, Roberto

    2012-01-01

    Cellulose waste biomass is the most abundant and attractive substrate for “biorefinery strategies” that are aimed to produce high-value products (e.g. solvents, fuels, building blocks) by economically and environmentally sustainable fermentation processes. However, cellulose is highly recalcitrant to biodegradation and its conversion by biotechnological strategies currently requires economically inefficient multistep industrial processes. The need for dedicated cellulase production continues to be a major constraint to cost-effective processing of cellulosic biomass. Research efforts have been aimed at developing recombinant microorganisms with suitable characteristics for single step biomass fermentation (consolidated bioprocessing, CBP). Two paradigms have been applied for such, so far unsuccessful, attempts: a) “native cellulolytic strategies”, aimed at conferring high-value product properties to natural cellulolytic microorganisms; b) “recombinant cellulolytic strategies”, aimed to confer cellulolytic ability to microorganisms exhibiting high product yields and titers. By starting from the description of natural enzyme systems for plant biomass degradation and natural metabolic pathways for some of the most valuable product (i.e. butanol, ethanol, and hydrogen) biosynthesis, this review describes state-of-the-art bottlenecks and solutions for the development of recombinant microbial strains for cellulosic biofuel CBP by metabolic engineering. Complexed cellulases (i.e. cellulosomes) benefit from stronger proximity effects and show enhanced synergy on insoluble substrates (i.e. crystalline cellulose) with respect to free enzymes. For this reason, special attention was held on strategies involving cellulosome/designer cellulosome-bearing recombinant microorganisms. PMID:24688667

  2. Development of microorganisms for cellulose-biofuel consolidated bioprocessings: metabolic engineers' tricks.

    PubMed

    Mazzoli, Roberto

    2012-01-01

    Cellulose waste biomass is the most abundant and attractive substrate for "biorefinery strategies" that are aimed to produce high-value products (e.g. solvents, fuels, building blocks) by economically and environmentally sustainable fermentation processes. However, cellulose is highly recalcitrant to biodegradation and its conversion by biotechnological strategies currently requires economically inefficient multistep industrial processes. The need for dedicated cellulase production continues to be a major constraint to cost-effective processing of cellulosic biomass. Research efforts have been aimed at developing recombinant microorganisms with suitable characteristics for single step biomass fermentation (consolidated bioprocessing, CBP). Two paradigms have been applied for such, so far unsuccessful, attempts: a) "native cellulolytic strategies", aimed at conferring high-value product properties to natural cellulolytic microorganisms; b) "recombinant cellulolytic strategies", aimed to confer cellulolytic ability to microorganisms exhibiting high product yields and titers. By starting from the description of natural enzyme systems for plant biomass degradation and natural metabolic pathways for some of the most valuable product (i.e. butanol, ethanol, and hydrogen) biosynthesis, this review describes state-of-the-art bottlenecks and solutions for the development of recombinant microbial strains for cellulosic biofuel CBP by metabolic engineering. Complexed cellulases (i.e. cellulosomes) benefit from stronger proximity effects and show enhanced synergy on insoluble substrates (i.e. crystalline cellulose) with respect to free enzymes. For this reason, special attention was held on strategies involving cellulosome/designer cellulosome-bearing recombinant microorganisms.

  3. Microorganism billiards

    NASA Astrophysics Data System (ADS)

    Spagnolie, Saverio E.; Wahl, Colin; Lukasik, Joseph; Thiffeault, Jean-Luc

    2017-02-01

    Recent experiments and numerical simulations have shown that certain types of microorganisms "reflect" off of a flat surface at a critical angle of departure, independent of the angle of incidence. The nature of the reflection may be active (cell and flagellar contact with the surface) or passive (hydrodynamic) interactions. We explore the billiard-like motion of a body with this empirical reflection law inside a regular polygon and show that the dynamics can settle on a stable periodic orbit or can be chaotic, depending on the swimmer's departure angle and the domain geometry. The dynamics are often found to be robust to the introduction of weak random fluctuations. The Lyapunov exponent of swimmer trajectories can be positive or negative, can have extremal values, and can have discontinuities depending on the degree of the polygon. A passive sorting device is proposed that traps swimmers of different departure angles into separate bins. We also study the external problem of a microorganism swimming in a patterned environment of square obstacles, where the departure angle dictates the possibility of trapping or diffusive trajectories.

  4. Human leukocytic pyrogen test for detection of pyrogenic material in growth hormone produced by recombinant Escherichia coli.

    PubMed Central

    Dinarello, C A; O'Connor, J V; LoPreste, G; Swift, R L

    1984-01-01

    Human growth hormone is biosynthetically produced in recombinant strains of Escherichia coli as methionyl human growth hormone (met-hGH). When purified from the bacterial culture, met-hGH is biologically active in established assays for growth hormone. Therefore, a phase I trial of met-hGH was carried out in healthy human adults; during the first trial, however, signs, symptoms, and clinical laboratory tests characteristic of an acute-phase response to pyrogenic agents was observed. Prior testing of the met-hGH preparation used in the phase I trial did not reveal evidence of toxicity, and the U.S. Pharmacopeial Convention rabbit pyrogen test, as well as the Limulus amoebocyte lysate (LAL) test, had not detected significant levels of exogenous pyrogens or endotoxin. In addition, standard inhibition studies with added endotoxin showed no inhibition by the LAL test. When this preparation of met-hGH was incubated with human blood mononuclear cells, leukocytic pyrogen (LP) was released into the supernatant medium, suggesting that the preparation contained pyrogenic material. Various lots of met-hGH based on different purification and formulating methods were tested by the human LP assay for contaminating pyrogens. The results of these tests aided in the identification of procedures for met-hGH preparations which did not induce LP in vitro. Thus, subsequent lots of met-hGH which had passed the LP test were used in repeat clinical studies, and no inflammatory or pyrogenic reactions were observed. When the LP test was used, experiments revealed that the original lot of met-hGH was contaminated with endotoxin which had not been detected in the LAL or rabbit pyrogen tests. Lyophilization in glycine-phosphate buffer had resulted in a 10- to 20-fold reduction of endotoxin reactivity in the LAL test and the U.S. Pharmacopeial Convention rabbit pyrogen test. These data provide a probable explanation for the negative result from the LAL and rabbit pyrogen test in the initial lot

  5. Recombination occurs within minutes of replication blockage by RTS1 producing restarted forks that are prone to collapse

    PubMed Central

    Nguyen, Michael O; Jalan, Manisha; Morrow, Carl A; Osman, Fekret; Whitby, Matthew C

    2015-01-01

    The completion of genome duplication during the cell cycle is threatened by the presence of replication fork barriers (RFBs). Following collision with a RFB, replication proteins can dissociate from the stalled fork (fork collapse) rendering it incapable of further DNA synthesis unless recombination intervenes to restart replication. We use time-lapse microscopy and genetic assays to show that recombination is initiated within ∼10 min of replication fork blockage at a site-specific barrier in fission yeast, leading to a restarted fork within ∼60 min, which is only prevented/curtailed by the arrival of the opposing replication fork. The restarted fork is susceptible to further collapse causing hyper-recombination downstream of the barrier. Surprisingly, in our system fork restart is unnecessary for maintaining cell viability. Seemingly, the risk of failing to complete replication prior to mitosis is sufficient to warrant the induction of recombination even though it can cause deleterious genetic change. DOI: http://dx.doi.org/10.7554/eLife.04539.001 PMID:25806683

  6. Uric acid in plants and microorganisms: Biological applications and genetics - A review.

    PubMed

    Hafez, Rehab M; Abdel-Rahman, Tahany M; Naguib, Rasha M

    2017-09-01

    Uric acid increased accumulation and/or reduced excretion in human bodies is closely related to pathogenesis of gout and hyperuricemia. It is highly affected by the high intake of food rich in purine. Uric acid is present in both higher plants and microorganisms with species dependent concentration. Urate-degrading enzymes are found both in plants and microorganisms but the mechanisms by which plant degrade uric acid was found to be different among them. Higher plants produce various metabolites which could inhibit xanthine oxidase and xanthine oxidoreductase, so prohibit the oxidation of hypoxanthine to xanthine then to uric acid in the purine metabolism. However, microorganisms produce group of degrading enzymes uricase, allantoinase, allantoicase and urease, which catalyze the degradation of uric acid to the ammonia. In humans, researchers found that several mutations caused a pseudogenization (silencing) of the uricase gene in ancestral apes which exist as an insoluble crystalloid in peroxisomes. This is in contrast to microorganisms in which uricases are soluble and exist either in cytoplasm or peroxisomes. Moreover, many recombinant uricases with higher activity than the wild type uricases could be induced successfully in many microorganisms. The present review deals with the occurrence of uric acid in plants and other organisms specially microorganisms in addition to the mechanisms by which plant extracts, metabolites and enzymes could reduce uric acid in blood. The genetic and genes encoding for uric acid in plants and microorganisms are also presented.

  7. Molecular farming of human cytokines and blood products from plants: challenges in biosynthesis and detection of plant-produced recombinant proteins.

    PubMed

    da Cunha, Nicolau B; Vianna, Giovanni R; da Almeida Lima, Thaina; Rech, Elíbio

    2014-01-01

    Plants have emerged as an attractive alternative to the traditional mammalian cell cultures or microbial cell-based systems system for the production of valuable recombinant proteins. Through recombinant DNA technology, plants can be engineered to produce large quantities of pharmaceuticals and industrial proteins of high quality at low costs. The recombinant production, by transgenic plants, of therapeutic proteins normally present in human plasma, such as cytokines, coagulation factors, anticoagulants, and immunoglobulins, represents a response to the ongoing challenges in meeting the demand for therapeutic proteins to treat serious inherited or acquired bleeding and immunological diseases. As the clinical utilization of fractionated plasma molecules is limited by high production costs, using recombinant biopharmaceuticals derived from plants represents a feasible alternative to provide efficient treatment. Plant-derived pharmaceuticals also reduce the potential risks to patients of infection with pathogens or unwanted immune responses due to immunogenic antigens. In this review, we summarize the recent advances in molecular farming of cytokines. We also examine the technological basis, upcoming challenges, and perspectives for the biosynthesis and detection of these molecules in different plant production platforms.

  8. Origin of the CMS gene locus in rapeseed cybrid mitochondria: active and inactive recombination produces the complex CMS gene region in the mitochondrial genomes of Brassicaceae.

    PubMed

    Oshima, Masao; Kikuchi, Rie; Imamura, Jun; Handa, Hirokazu

    2010-01-01

    CMS (cytoplasmic male sterile) rapeseed is produced by asymmetrical somatic cell fusion between the Brassica napus cv. Westar and the Raphanus sativus Kosena CMS line (Kosena radish). The CMS rapeseed contains a CMS gene, orf125, which is derived from Kosena radish. Our sequence analyses revealed that the orf125 region in CMS rapeseed originated from recombination between the orf125/orfB region and the nad1C/ccmFN1 region by way of a 63 bp repeat. A precise sequence comparison among the related sequences in CMS rapeseed, Kosena radish and normal rapeseed showed that the orf125 region in CMS rapeseed consisted of the Kosena orf125/orfB region and the rapeseed nad1C/ccmFN1 region, even though Kosena radish had both the orf125/orfB region and the nad1C/ccmFN1 region in its mitochondrial genome. We also identified three tandem repeat sequences in the regions surrounding orf125, including a 63 bp repeat, which were involved in several recombination events. Interestingly, differences in the recombination activity for each repeat sequence were observed, even though these sequences were located adjacent to each other in the mitochondrial genome. We report results indicating that recombination events within the mitochondrial genomes are regulated at the level of specific repeat sequences depending on the cellular environment.

  9. [Biotechnology using modified microorganisms].

    PubMed

    Deshayes, A F

    1992-11-01

    Few microorganisms, as compare to their high diversity, are used for human needs. They can produce molecules of interest, process fermentation, protect crops, treat wastes or clean environment. Molecular technics and genetic engineering are new tools offer to geneticists which breed microorganisms for years. Using them, it is now possible, theoretically, to introduce any gene in any organism. Some examples are given concerning genetic modifications in yeasts and lactic acid bacteria to optimize agrofood processes and to improve nutritive and flavour characteristics of fermented products like bread, beer, wine, cheese, meat, vegetable juices... In spite of scientific and industrial interest of the new technologies, limiting factors can explain that genetically modified microorganisms are not routinely used in agrofood yet. First, risks assessment on human health and environment are still in debate, but their is a consensus, within the scientific community, to consider that new characteristics of improved microorganisms are more important than the technics used for their construction. Second, regulations turn out to impose constraints susceptible to discourage technological innovations. At least, the public perception about the new technologies appears, actually, as the major factor to limit their development.

  10. Genes encoding homologous antigens in taeniid cestode parasites: Implications for development of recombinant vaccines produced in Escherichia coli.

    PubMed

    Gauci, Charles; Lightowlers, Marshall W

    2013-01-01

    Recombinant vaccine antigens are being evaluated for their ability to protect livestock animals against cysticercosis and related parasitic infections. Practical use of some of these vaccines is expected to reduce parasite transmission, leading to a reduction in the incidence of neurocysticercosis and hydatid disease in humans. We recently showed that an antigen (TSOL16), expressed in Escherichia coli, confers high levels of protection against Taenia solium cysticercosis in pigs, which provides a strategy for control of T. solium parasite transmission. Here, we discuss the characteristics of this antigen that may affect the utility of TSOL16 and related antigens for development as recombinant vaccines. We also report that genes encoding antigens closely related to TSOL16 from T. solium also occur in other related species of parasites. These highly homologous antigens have the potential to be used as vaccines and may provide protection against related species of Taenia that cause infection in other hosts.

  11. Identifying the risk of producing aneuploids using meiotic recombination genes as biomarkers: A copy number variation approach

    PubMed Central

    Suresh, Raviraj V.; Lingaiah, Kusuma; Veerappa, Avinash M.; Ramachandra, Nallur B.

    2017-01-01

    Background & objectives: Aneuploids are the most common chromosomal abnormality in liveborns and are usually the result of non-disjunction (NDJ) in meiosis. Copy number variations (CNVs) are large structural variations affecting the human genome. CNVs influence critical genes involved in causing NDJ by altering their copy number which affects the clinical outcome. In this study influence of CNVs on critical meiotic recombination was examined using new computational technologies to assess their role in causing aneuploidy. Methods: This investigation was based on the analysis of 12 random normal populations consisting of 1714 individuals for aneuploid causing genes under CNV effect. To examine the effect of CNVs on genes causing aneuploidy, meiotic recombination genes were analyzed using EnrichR, WebGestalt and Ingenuity Pathway Analysis (IPA). Results: Forty three NDJ genes were found under CNV burden; IPA (Ingenuity Pathway Analysis) and KEGG (Kyoto Encyclopedia of Genes and Genomes) pathway analysis of CNV in meiotic recombination genes revealed a significant role of breast cancer gene 1, amyloid protein precursor, mitogen-activated protein kinase and nerve growth factor as key molecular players involved in causing aneuploidy. Interaction between these genes with other CNV-overlapping genes involved in cell cycle, recombination and meiosis might lead to increased incidences of aneuploidy. Interpretation & conclusions: The findings of this study implied that the effect of CNVs on normal genome contributed in amplifying the occurrences of chromosomal aneuploidies. The normal individuals consisting of variations in the susceptible genes causing aneuploids in the population remain undetected until the disorder genes express in the succeeding generations. PMID:28574013

  12. Fusion of the Dhfr/Mtx and IR/MAR gene amplification methods produces a rapid and efficient method for stable recombinant protein production.

    PubMed

    Noguchi, Chiemi; Araki, Yoshio; Miki, Daisuke; Shimizu, Noriaki

    2012-01-01

    Amplification of the dihydrofolate reductase gene (Dhfr) by methotrexate (Mtx) exposure is commonly used for recombinant protein expression in Chinese hamster ovary (CHO) cells. However, this method is both time- and labor-intensive, and the high-producing cells that are generated are frequently unstable in culture. Another gene amplification method is based on using a plasmid bearing a mammalian replication initiation region (IR) and a matrix attachment region (MAR), which result in the spontaneous initiation of gene amplification in transfected cells. The IR/MAR and Dhfr/Mtx methods of gene amplification are based on entirely different principles. In this study, we combine these two methods to yield a novel method, termed the IR/MAR-Dhfr fusion method, which was used to express three proteins, the Fc receptor, GFP, and recombinant antibody. The fusion method resulted in a dramatic increase in expression of all three proteins in two CHO sub-lines, DXB-11, and DG44. The IR/MAR-Dhfr fusion amplified the genes rapidly and efficiently, and produced larger amounts of antibody than the Dhfr/Mtx or IR/MAR methods alone. While the amplified structure produced by the Dhfr/Mtx method was highly unstable, and the antibody production rate rapidly decreased with the culture time of the cells, the IR/MAR-Dhfr fusion method resulted in stable amplification and generated clonal cells that produced large amounts of antibody protein over a long period of time. In summary, the novel IR/MAR-Dhfr fusion method enables isolation of stable cells that produce larger amounts of a target recombinant protein more rapidly and easily than either the Dhfr/Mtx or IR/MAR methods alone.

  13. Fusion of the Dhfr/Mtx and IR/MAR Gene Amplification Methods Produces a Rapid and Efficient Method for Stable Recombinant Protein Production

    PubMed Central

    Miki, Daisuke; Shimizu, Noriaki

    2012-01-01

    Amplification of the dihydrofolate reductase gene (Dhfr) by methotrexate (Mtx) exposure is commonly used for recombinant protein expression in Chinese hamster ovary (CHO) cells. However, this method is both time- and labor-intensive, and the high-producing cells that are generated are frequently unstable in culture. Another gene amplification method is based on using a plasmid bearing a mammalian replication initiation region (IR) and a matrix attachment region (MAR), which result in the spontaneous initiation of gene amplification in transfected cells. The IR/MAR and Dhfr/Mtx methods of gene amplification are based on entirely different principles. In this study, we combine these two methods to yield a novel method, termed the IR/MAR-Dhfr fusion method, which was used to express three proteins, the Fc receptor, GFP, and recombinant antibody. The fusion method resulted in a dramatic increase in expression of all three proteins in two CHO sub-lines, DXB-11, and DG44. The IR/MAR-Dhfr fusion amplified the genes rapidly and efficiently, and produced larger amounts of antibody than the Dhfr/Mtx or IR/MAR methods alone. While the amplified structure produced by the Dhfr/Mtx method was highly unstable, and the antibody production rate rapidly decreased with the culture time of the cells, the IR/MAR-Dhfr fusion method resulted in stable amplification and generated clonal cells that produced large amounts of antibody protein over a long period of time. In summary, the novel IR/MAR-Dhfr fusion method enables isolation of stable cells that produce larger amounts of a target recombinant protein more rapidly and easily than either the Dhfr/Mtx or IR/MAR methods alone. PMID:23300841

  14. Comparative analysis of plant-produced, recombinant dimeric IgA against cell wall β-glucan of pathogenic fungi.

    PubMed

    Capodicasa, Cristina; Catellani, Marcello; Moscetti, Ilaria; Bromuro, Carla; Chiani, Paola; Torosantucci, Antonella; Benvenuto, Eugenio

    2017-08-19

    Immunoglobulins A (IgA) are crucially involved in protection of human mucosal surfaces from microbial pathogens. In this work, we devised and expressed in plants recombinant chimeric antifungal antibodies (Abs) of isotype A (IgA1, IgA2, and scFvFcA1), derived from a murine mAb directed to the fungal cell wall polysaccharide β-glucan which had proven able to confer protection against multiple pathogenic fungi. All recombinant IgA (rIgA) were expressed and correctly assembled in dimeric form in plants and evaluated for yield, antigen-binding efficiency and antifungal properties in vitro, in comparison with a chimeric IgG1 version. Production yields and binding efficiency to purified β-glucans showed significant variations not only between Abs of different isotypes but also between the different IgA formats. Moreover, only the dimeric IgA1 was able to strongly bind cells of the fungal pathogen Candida albicans and to restrain its adhesion to human epithelial cells. Our data indicate that IgG to IgA switch and differences in molecular structure among different rIgA formats can impact expression in plant and biological activity of anti-β-glucans Abs and provide new insights for the design of recombinant IgA as anti-infective immunotherapeutics, whose potential is still poorly investigated. © 2017 Wiley Periodicals, Inc.

  15. Purification of inclusion bodies using PEG precipitation under denaturing conditions to produce recombinant therapeutic proteins from Escherichia coli.

    PubMed

    Chen, Huanhuan; Li, Ninghuan; Xie, Yueqing; Jiang, Hua; Yang, Xiaoyi; Cagliero, Cedric; Shi, Siwei; Zhu, Chencen; Luo, Han; Chen, Junsheng; Zhang, Lei; Zhao, Menglin; Feng, Lei; Lu, Huili; Zhu, Jianwei

    2017-07-01

    It has been documented that the purification of inclusion bodies from Escherichia coli by size exclusion chromatography (SEC) may benefit subsequent refolding and recovery of recombinant proteins. However, loading volume and the high cost of the column limits its application in large-scale manufacturing of biopharmaceutical proteins. We report a novel process using polyethylene glycol (PEG) precipitation under denaturing conditions to replace SEC for rapid purification of inclusion bodies containing recombinant therapeutic proteins. Using recombinant human interleukin 15 (rhIL-15) as an example, inclusion bodies of rhIL-15 were solubilized in 7 M guanidine hydrochloride, and rhIL-15 was precipitated by the addition of PEG 6000. A final concentration of 5% (w/v) PEG 6000 was found to be optimal to precipitate target proteins and enhance recovery and purity. Compared to the previously reported S-200 size exclusion purification method, PEG precipitation was easier to scale up and achieved the same protein yields and quality of the product. PEG precipitation also reduced manufacturing time by about 50 and 95% of material costs. After refolding and further purification, the rhIL-15 product was highly pure and demonstrated a comparable bioactivity with a rhIL-15 reference standard. Our studies demonstrated that PEG precipitation of inclusion bodies under denaturing conditions holds significant potential as a manufacturing process for biopharmaceuticals from E. coli protein expression systems.

  16. Characterization and downstream mannose phosphorylation of human recombinant α-L-iduronidase produced in Arabidopsis complex glycan-deficient (cgl) seeds.

    PubMed

    He, Xu; Pierce, Owen; Haselhorst, Thomas; von Itzstein, Mark; Kolarich, Daniel; Packer, Nicolle H; Gloster, Tracey M; Vocadlo, David J; Qian, Yi; Brooks, Doug; Kermode, Allison R

    2013-12-01

    Mucopolysaccharidosis (MPS) I is a lysosomal storage disease caused by a deficiency of α-L-iduronidase (IDUA) (EC 3.2.1.76); enzyme replacement therapy is the conventional treatment for this genetic disease. Arabidopsis cgl mutants are characterized by a deficiency of the activity of N-acetylglucosaminyl transferase I (EC 2.4.1.101), the first enzyme in the pathway of hybrid and complex N-glycan biosynthesis. To develop a seed-based platform for the production of recombinant IDUA for potential treatment of MPS I, cgl mutant seeds were generated to express human IDUA at high yields and to avoid maturation of the N-linked glycans on the recombinant human enzyme. Enzyme kinetic data showed that cgl-IDUA has similar enzymatic properties to the commercial recombinant IDUA derived from cultured Chinese hamster ovary (CHO) cells (Aldurazyme™). The N-glycan profile showed that cgl-derived IDUA contained predominantly high-mannose-type N-glycans (94.5%), and the residual complex/hybrid N-glycan-containing enzyme was efficiently removed by an additional affinity chromatography step. Furthermore, purified cgl-IDUA was amenable to sequential in vitro processing by soluble recombinant forms of the two enzymes that mediate the addition of the mannose-6-phosphate (M6P) tag in mammalian cells-UDP-GlcNAc:lysosomal enzyme N-acetylglucosamine (GlcNAc)-1-phosphotransferase-and GlcNAc-1-phosphodiester α-N-acetylglucosaminidase (the 'uncovering enzyme'). Arabidopsis seeds provide an alternative system for producing recombinant lysosomal enzymes for enzyme replacement therapy; the purified enzymes can be subjected to downstream processing to create the M6P, a recognition marker essential for efficient receptor-mediated uptake into lysosomes of human cells.

  17. Production of Resveratrol in Recombinant Microorganisms

    PubMed Central

    Beekwilder, Jules; Wolswinkel, Rianne; Jonker, Harry; Hall, Robert; de Vos, C. H. Ric; Bovy, Arnaud

    2006-01-01

    Resveratrol production in Saccharomyces cerevisiae was compared to that in Escherichia coli. In both systems, 4-coumarate:coenzyme A ligase from tobacco and stilbene synthase from grapes were expressed. When p-coumaric acid was used as the precursor, resveratrol accumulations in the culture medium were observed to be comparable in E. coli (16 mg/liter) and yeast (6 mg/liter). PMID:16885328

  18. Metabolic engineering towards biotechnological production of carotenoids in microorganisms.

    PubMed

    Lee, P C; Schmidt-Dannert, C

    2002-10-01

    Carotenoids are important natural pigments produced by many microorganisms and plants. Traditionally, carotenoids have been used in the feed, food and nutraceutical industries. The recent discoveries of health-related beneficial properties attributed to carotenoids have spurred great interest in the production of structurally diverse carotenoids for pharmaceutical applications. The availability of a considerable number of microbial and plant carotenoid genes that can be functionally expressed in heterologous hosts has opened ways for the production of diverse carotenoid compounds in heterologous systems. In this review, we will describe the recent progress made in metabolic engineering of non-carotenogenic microorganisms for improved carotenoid productivity. In addition, we will discuss the application of combinatorial and evolutionary strategies to carotenoid pathway engineering to broaden the diversity of carotenoid structures synthesized in recombinant hosts.

  19. Temperature effects on product-quality-related enzymes in batch CHO cell cultures producing recombinant tPA.

    PubMed

    Clark, Kevin J R; Chaplin, Frank W R; Harcum, Sarah W

    2004-01-01

    Culture conditions that affect product quality are important to the successful operation and optimization of bioreactors. Previous studies have demonstrated that enzymes, such as proteases and sialidases, accumulate in batch bioreactors. These enzymes are known to be detrimental to the quality of recombinant glycoproteins. Bioreactor temperature has been used to control cell growth and recombinant protein production rates. However, the effect of culture temperature on the production of proteases and sialidases has not been investigated. In this study, Chinese hamster ovary cells were cultured with a temperature profile that decreased from 37 to 34 degrees C over 8 days and with a constant temperature of 37 degrees C. Analysis of extracellular protease activity indicated that two major proteases were present (50 and 69 kDa). The 50 kDa protease activity decreased similarly with time for both culture conditions. The 69 kDa protease activity increased with time for both culture conditions. The constant-temperature cultures had significantly lower 69 kDa protease levels compared to the ramped-temperature cultures in the early stationary phase. Intracellular sialidase activity was present in both cultures. The intracellular sialidase activity increased dramatically for both culture conditions immediately after the cells were inoculated into fresh medium. The initial peak in intracellular sialidase activity was followed by a first-order decay. The intracellular sialidase activities for the two culture conditions were not significantly different. The production of recombinant tissue type plasminogen activator was not significantly different for the two culture conditions. Thus, the previously hypothesized advantages that lower culture temperatures have reduced protease activity and improved productivity do not appear to be universal.

  20. DNA sequences, recombinant DNA molecules and processes for producing bovine growth hormone-like polypeptides in high yield

    SciTech Connect

    Buell, G.N.

    1987-09-15

    This patent describes a process for increasing the yield of a bovine growth hormone-like polypeptide to at least 100 times that of a bovine growth hormone-like polypeptide encoded by a DNA sequence. The process comprises the steps of culturing a host transformed with a recombinant DNA molecule comprising DNA sequence encoding a Met ..lambda.. or ..lambda.. bovine growth hormone-like polypetide operatively linked to an expression control sequence. The ..lambda.. is an amino terminal deletion from the amino acid sequence of mature bovine growth hormone.

  1. Transgenic cows that produce recombinant human lactoferrin in milk are not protected from experimental Escherichia coli intramammary infection.

    PubMed

    Hyvönen, P; Suojala, L; Orro, T; Haaranen, J; Simola, O; Røntved, C; Pyörälä, S

    2006-11-01

    This is the first study describing an experimental mastitis model using transgenic cows expressing recombinant human lactoferrin (rhLf) in their milk. The aim of the study was to investigate the concentrations in milk and protective effects of bovine and recombinant human lactoferrin in experimental Escherichia coli mastitis. Experimental intramammary infection was induced in one udder quarter of seven first-lactating rhLf-transgenic cows and six normal cows, using an E. coli strain isolated from cows with clinical mastitis and known to be susceptible to Lf in vitro. Clinical signs were recorded during the experimental period, concentrations of human and bovine Lf and indicators of inflammation and bacterial counts were determined for milk, and concentrations of acute-phase proteins and tumor necrosis factor alpha were determined for sera and milk. Serum cortisol and blood hematological and biochemical parameters were also determined. Expression levels of rhLf in the milk of transgenic cows remained constant throughout the experiment (mean, 2.9 mg/ml). The high Lf concentrations in the milk of transgenic cows did not protect them from intramammary infection. All cows became infected and developed clinical mastitis. The rhLf-transgenic cows showed milder systemic signs and lower serum cortisol and haptoglobin concentrations than did controls. This may be explained by lipopolysaccharide-neutralizing and immunomodulatory effects of the high Lf concentrations in their milk. However, Lf does not seem to be a very efficient protein for genetic engineering to enhance the mastitis resistance of dairy cows.

  2. [Characterization of hepatitis C virus structural proteins and HCV-like particles produced in recombinant baculovirus infected insect cells].

    PubMed

    Belzhelarskaia, S N; Koroleva, N N; Popenko, V V; Drutsa, V L; Orlova, O V; Rubtsov, P M; Kochetkov, S N

    2010-01-01

    Three proteins, namely: "core" protein C and glycoproteins E1 and E2, are main structural proteins forming a hepatitis C vius (HCV) virion. The virus structure and assembly, a role of the structural proteins in virion morphogenesis remain unknown because of the lack of an efficient culture system for HCV to be grown in vitro. Using recombinant baculoviruses expressing HCV structural protein genes in insect cells the specific structural proteins at the level of 25-35% relative to a common cell protein content, heterodimers of the glcoproteins, and HCV-like particles have been obtained. It has been demonstrated that recombinant proteins C, E1, and E2 go through the posttranslation modification, the glycoproteins form the non-covalent heterodimer, and HCV-like particles are located in endoplasmatic reticulum membrains of infected cells. An ability of the expressed proteins for forming E1E2 dimers and HCV-like particles was used for studying the role of E1 protein glcosylation upon expression and processing of the glycoproteins.

  3. Co-cultivation of Aspergillus nidulans Recombinant Strains Produces an Enzymatic Cocktail as Alternative to Alkaline Sugarcane Bagasse Pretreatment

    PubMed Central

    Lima, Matheus S.; Damasio, André R. de L.; Crnkovic, Paula M.; Pinto, Marcelo R.; da Silva, Ana M.; da Silva, Jean C. R.; Segato, Fernando; de Lucas, Rosymar C.; Jorge, João A.; Polizeli, Maria de L. T. de M.

    2016-01-01

    Plant materials represent a strategic energy source because they can give rise to sustainable biofuels through the fermentation of their carbohydrates. A clear example of a plant-derived biofuel resource is the sugar cane bagasse exhibiting 60–80% of fermentable sugars in its composition. However, the current methods of plant bioconversion employ severe and harmful chemical/physical pretreatments raising biofuel cost production and environmental degradation. Replacing these methods with co-cultivated enzymatic cocktails is an alternative. Here we propose a pretreatment for sugarcane bagasse using a multi-enzymatic cocktail from the co-cultivation of four Aspergillus nidulans recombinant strains. The co-cultivation resulted in the simultaneous production of GH51 arabinofuranosidase (AbfA), GH11 endo-1,4-xylanase (XlnA), GH43 endo-1,5-arabinanase (AbnA) and GH12 xyloglucan specific endo-β-1,4-glucanase (XegA). This core set of recombinant enzymes was more efficient than the alternative alkaline method in maintaining the cellulose integrity and exposing this cellulose to the following saccharification process. Thermogravimetric and differential thermal analysis revealed residual byproducts on the alkali pretreated biomass, which were not found in the enzymatic pretreatment. Therefore, the enzymatic pretreatment was residue-free and seemed to be more efficient than the applied alkaline method, which makes it suitable for bioethanol production. PMID:27199917

  4. Co-cultivation of Aspergillus nidulans Recombinant Strains Produces an Enzymatic Cocktail as Alternative to Alkaline Sugarcane Bagasse Pretreatment.

    PubMed

    Lima, Matheus S; Damasio, André R de L; Crnkovic, Paula M; Pinto, Marcelo R; da Silva, Ana M; da Silva, Jean C R; Segato, Fernando; de Lucas, Rosymar C; Jorge, João A; Polizeli, Maria de L T de M

    2016-01-01

    Plant materials represent a strategic energy source because they can give rise to sustainable biofuels through the fermentation of their carbohydrates. A clear example of a plant-derived biofuel resource is the sugar cane bagasse exhibiting 60-80% of fermentable sugars in its composition. However, the current methods of plant bioconversion employ severe and harmful chemical/physical pretreatments raising biofuel cost production and environmental degradation. Replacing these methods with co-cultivated enzymatic cocktails is an alternative. Here we propose a pretreatment for sugarcane bagasse using a multi-enzymatic cocktail from the co-cultivation of four Aspergillus nidulans recombinant strains. The co-cultivation resulted in the simultaneous production of GH51 arabinofuranosidase (AbfA), GH11 endo-1,4-xylanase (XlnA), GH43 endo-1,5-arabinanase (AbnA) and GH12 xyloglucan specific endo-β-1,4-glucanase (XegA). This core set of recombinant enzymes was more efficient than the alternative alkaline method in maintaining the cellulose integrity and exposing this cellulose to the following saccharification process. Thermogravimetric and differential thermal analysis revealed residual byproducts on the alkali pretreated biomass, which were not found in the enzymatic pretreatment. Therefore, the enzymatic pretreatment was residue-free and seemed to be more efficient than the applied alkaline method, which makes it suitable for bioethanol production.

  5. Specific features of recombination processes in CdTe films produced in different temperature conditions of growth and subsequent annealing

    SciTech Connect

    Ermolovich, I. B.; Milenin, V. V.; Red'ko, R. A. Red'ko, S. M.

    2009-08-15

    The steady-state and kinetic characteristics of photoconductivity and photoluminescence and the thermally stimulated conductivity spectra of the GdTe layers deposited by vacuum evaporation onto heated substrates are studied in relation to the substrate temperature. The measurements are carried out at temperatures, illuminations, and wavelengths ranging from 4.2 to 400 K, from 10{sup 10} to 10{sup 23} photon/cm{sup 2}, and from0.4 to 2.5 {mu}m, respectively. A certain optimal range of substrate temperatures T{sub s} {approx} 450-550 deg. C, at which the as-prepared layers exhibit a high resistivity, a high photosensitivity, and the best structural quality, is established. In the spectra of these layers, a new luminescence band at hv{sub m} = 1.09 eV is observed along with the known photoluminescence band at hv{sub m} = 1.42 eV. It is established that this new band is due to intracenter transitions rather than recombination transitions. The nature of radiative recombination centers in the layers is discussed. It is suggested that the d electrons of cations can be involved in the formation of chemical bonds of local centers in CdTe.

  6. Establishment of a large-scale purification procedure for purified recombinant bovine interferon-tau produced by a silkworm-baculovirus gene expression system.

    PubMed

    Nagaya, Hidekazu; Kanaya, Toshimichi; Kaki, Hiroki; Tobita, Yoneko; Takahashi, Masashi; Takahashi, Hitomi; Yokomizo, Yuichi; Inumaru, Shigeki

    2004-11-01

    We developed a procedure for the large-scale purification of bovine interferon-tau (boIFN-tau) by means of a silkworm-baculovirus gene expression system. Recombinant boIFN-tau (rboIFN-tau) was efficiently produced in the silkworm infected with boIFN-tau cDNA recombinant baculovirus and accumulated in the haemolymph. To establish a purification method suitable for mass production, we tried three crude purification methods, namely, an acidification and neutralization treatment (ANT), silica gel column chromatography (SGCC), and Blue sepharose column chromatography (BSCC) with a combination of Q-sepharose (QSC) and chelating sepharose column chromatographies (CSCC). As a result, the acidification and neutralization treatment was found to be the most efficient and cost effective. With this combination, we obtained 91% pure products. To confirm the applicability of the procedure for mass production, we inoculated 100 silkworms with the recombinant virus, and recovered about 4.55 mg (1.26 x 10(8)U/mg) of 91% pure rboIFN-tau by means of a combination of the ANT, followed by QSC and CSCC.

  7. Recombinant Scorpine Produced Using SUMO Fusion Partner in Escherichia coli Has the Activities against Clinically Isolated Bacteria and Inhibits the Plasmodium falciparum Parasitemia In Vitro

    PubMed Central

    Gu, Yaping; Zhou, Huayun; Cao, Jun; Gao, Qi

    2014-01-01

    Scorpine, a small cationic peptide from the venom of Pandinus imperator, which has been shown to have anti-bacterial and anti-plasmodial activities, has potential important applications in the pharmaceutical industries. However, the isolation of scorpine from natural sources is inefficient and time-consuming. Here, we first report the expression and purification of recombinant scorpine in Escherichia coli, using small ubiquitin-related modifier (SUMO) fusion partner. The fusion protein was expressed in soluble form in E. coli, and expression was verified by SDS-PAGE and western blotting analysis. The fusion protein was purified to 90% purity by nickel–nitrilotriacetic acid (Ni2+–NTA) resin chromatography. After the SUMO-scorpine fusion protein was cleaved by the SUMO protease, the cleaved sample was reapplied to a Ni2+–NTA column. Tricine/SDS-PAGE gel results indicated that Scorpine had been purified successfully to more than 95% purity. The recombinantly expressed Scorpine showed anti-bacterial activity against two standard bacteria including Staphylococcus aureus ATCC 29213 and Acinetobacter baumannii ATCC 19606, and clinically isolated bacteria including S. aureus S, S. aureus R, A. baumannii S, and A. baumannii R. It also produced 100% reduction in Plasmodium falciparum parasitemia in vitro. Thus, the expression strategy presented in this study allowed convenient high yield and easy purification of recombinant Scorpine for pharmaceutical applications in the future. PMID:25068263

  8. Recombinant Adenovirus Delivery of Calreticulin–ESAT-6 Produces an Antigen-Specific Immune Response but no Protection Against a Mycobacterium Tuberculosis Challenge

    PubMed Central

    Esparza-González, S. C.; Troy, A.; Troudt, J.; Loera-Arias, M. J.; Villatoro-Hernández, J.; Torres-López, E.; Ancer-Rodríguez, J.; Gutiérrez-Puente, Y.; Muñoz-Maldonado, G.; Saucedo-Cárdenas, O.; Montes-de-Oca-Luna, R.; Izzo, A.

    2015-01-01

    Bacillus Calmette–Guerin (BCG) has failed to efficaciously control the worldwide spread of the disease. New vaccine development targets virulence antigens of Mycobacterium tuberculosis that are deleted in Mycobacterium bovis BCG. Immunization with ESAT-6 and CFP10 provides protection against M. tuberculosis in a murine infection model. Further, previous studies have shown that calreticulin increases the cell-mediated immune responses to antigens. Therefore, to test whether calreticulin enhances the immune response against M. tuberculosis antigens, we fused ESAT-6 to calreticulin and constructed a recombinant replication-deficient adenovirus to express the resulting fusion protein (AdCRT–ESAT-6). The adjuvant effect of calreticulin was assayed by measuring cytokine responses specific to ESAT-6. Recombinant adenovirus expressing the fusion protein produced higher levels of interferon-γ and tumour necrosis factor-α in response to ESAT-6. This immune response was not improved by the addition of CFP-10 to the CRT-ESAT-6 fusion protein (AdCRT–ESAT-6–CFP10). Mice immunized with these recombinant adenoviruses did not decrease the mycobacterial burden after low-dose aerosol infection with M. tuberculosis. We conclude that calreticulin can be used as an adjuvant to enhance the immune response against mycobacterial antigens, but it is not enough to protect against tuberculosis. PMID:22010821

  9. Standardization of allergen products: 2. Detailed characterization of GMP-produced recombinant Phl p 5.0109 as European Pharmacopoeia reference standard.

    PubMed

    Himly, M; Nandy, A; Kahlert, H; Thilker, M; Steiner, M; Briza, P; Neubauer, A; Klysner, S; van Ree, R; Buchheit, K-H; Vieths, S; Ferreira, F

    2016-04-01

    The Biological Standardization Programme of the European Directorate for Quality of Medicines and Healthcare (EDQM) aims at the establishment of well-characterized reference standards based on recombinant allergens and validated assays for the quantification of major allergen content. The objective of this study was to examine the detailed physicochemical and immunological characterization of recombinant Phl p 5.0109, the second available allergen reference standard. Recombinant Phl p 5.0109 PP5ar06007 was produced under GMP conditions and analyzed by an array of physicochemical and immunological methods for identity, quantity, homogeneity, and folding stability in bulk solution, as well as thermal denaturation, aggregation state, and biological activity when formulated for long-time storage. PP5ar06007 revealed as a highly homogeneous, monomeric, well-folded preparation of rPhl p 5.0109, as documented by mass spectrometry, SDS-PAGE, isoelectric focusing, size-exclusion chromatography with light scattering, circular dichroism, and infrared spectroscopy. Upon storage at +4°C, PP5ar06007 retained the monomeric state for at least 2 months. A protein quantity of 1.56 ± 0.03 mg/ml was determined by amino acid analysis in PP5ar06007, and its biological activity was shown to be comparable to natural Phl p 5 in terms of basophil activation and T-cell reactivity. Recombinant Phl p 5.0109 PP5ar06007 was characterized extensively at the physicochemical and immunological level. It revealed to be a highly stable, monomeric, and immunologically equivalent of its natural counterpart. PP5ar06007 is now available as European Pharmacopoeia allergen reference standard for grass pollen products. © 2015 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

  10. Development of a mixed feed strategy for a recombinant Pichia pastoris strain producing with a de-repression promoter.

    PubMed

    Capone, Simona; Horvat, Jernej; Herwig, Christoph; Spadiut, Oliver

    2015-07-10

    Recombinant protein production in the yeast Pichia pastoris is usually based on the alcohol oxidase promoters pAOX1 and pAOX2, which are regulated by methanol and strongly repressed by other C-sources, like glycerol and glucose. However, the use of methanol brings several disadvantages, which is why current trends in bioprocess development with P. pastoris are focussing on minimizing the required amount of methanol or even avoid its employment. In this respect novel promoter systems which do not rely on methanol have been investigated and promoter variants were designed to fine-tune gene expression. Amongst these novel promoter systems, mutated AOX promoters, which are regulated by available carbon source concentration (so-called de-repressed promoters), are currently raising attention. However, the main disadvantage of such a production system is that expression and growth usually cannot happen concomitantly resulting in low space-time-yields. Here we show the development of a mixed-feed strategy for an industrial recombinant P. pastoris de-repression strain aiming at increased productivity and maximum space-time-yield. By doing dynamic experiments we determined a ratio between the specific substrate uptake rates of glycerol and sorbitol allowing a more than 2-fold increased productivity compared to the conventional single substrate de-repression strategy. Based on our results we recommend adjusting q(s glycerol) = 0.04 g g(-1) h(-1) and q(s sorbitol) = 0.055 g g(-1) h(-1) to obtain highest productivity with a P. pastoris de-repression strain. Our methodological approach of designing mixed-feed strategies based on physiological strain characterization using dynamic experiments proved to be beneficial.

  11. Production of 3-hydroxypropionic acid from glycerol by recombinant Klebsiella pneumoniae ΔdhaTΔyqhD which can produce vitamin B₁₂ naturally.

    PubMed

    Ashok, Somasundar; Sankaranarayanan, Mugesh; Ko, Yeounjoo; Jae, Kyeung-Eun; Ainala, Satish Kumar; Kumar, Vinod; Park, Sunghoon

    2013-02-01

    3-Hydroxypropionic acid (3-HP) is an important platform chemical that can be used to synthesize a range of chemical compounds. A previous study demonstrated that recombinant Escherichia coli stains can produce 3-HP from glycerol in the presence of vitamin B₁₂ (coenzyme B₁₂), when overexpressed with a coenzyme B₁₂-dependent glycerol dehydratase (DhaB) and an aldehyde dehydrogenase. The present study examined the production of 3-HP in recombinant Klebsiella pneumoniae strains, which naturally synthesizes vitamin B₁₂ and does not require supplementation of the expensive vitamin. The NAD⁺-dependent gamma-glutamyl-gamma-aminobutyraldehyde dehydrogenase (PuuC) of K. pneumoniae alone or with its DhaB was overexpressed homologously, and two major oxidoreductases, DhaT and YqhD, were disrupted. Without vitamin B₁₂ addition, the recombinant K. pneumoniae ΔdhaTΔyqhD overexpressing PuuC could produce ∼3.8 g/L 3-HP in 12 h of flask culture. However, this was possible only under the appropriate aeration conditions; 1,3-propanediol (1,3-PDO) (instead of 3-HP) was mainly produced when aeration was insufficient, whereas a very small amount of both 3-HP and 1,3-PDO were produced when aeration was too high. The production of a small amount of 3-HP under improper aeration conditions was attributed to either slow NAD⁺ regeneration (under low aeration) or reduced vitamin B₁₂ synthesis (under high aeration). In a glycerol fed-batch bioreactor experiment under a constant DO of 5%, the strain, K. pneumoniae ΔdhaTΔyqhD, overexpressing both PuuC and DhaB could produce >28 g/L 3-HP in 48 h with a yield of >40% on glycerol. Only small amount of 3-HP was produced when cultivation was carried out at a constant aeration of 1 vvm or constant 10% DO. These results show that K. pneumoniae is potentially useful for the production of 3-HP in an economical culture medium that does not require vitamin B₁₂. The results also suggest that the aeration

  12. Efficient selection of high-producing subclones during gene amplification of recombinant Chinese hamster ovary cells by flow cytometry and cell sorting.

    PubMed

    Borth, N; Zeyda, M; Kunert, R; Katinger, H

    The screening procedure for high-producing cell lines is extremely time- and labor-intensive and costly, and is at present guided by an empirical approach based on individual experience. Flow cytometry and cell sorting, with its ability to analyze and separate single cells, an ideal method in the selection of such rare cells. The isolation of recombinant cell lines is especially difficult due to repeated gene amplification, which introduces high mutational variation into the population. We have established and evaluated a modification of a previous method that traps secreted product on the surface of the secreting cell, thus allowing direct analysis of single cell specific production rates. This method was used to select for high-producing subclones of a recombinant Chinese hamster ovary (CHO) cell line producing a human antibody against HIV-1 by repeated rounds of gene amplification and cell sorting. This cell line has been amplified in previous investigations, so that the amount of work and testing required by traditional methods can be compared with the protocol described herein. Forty-five 96-well plates were necessary to obtain a high-producing subclone by limited dilution methods, whereas only five plates were required when cell sorting was used. The specific production rate of the best clone obtained by sorting, however, was five times that of the clone obtained by traditional methods. In contrast to the clones obtained by limited dilution, which consisted of several populations of low- and high-producing cells even at high methotrexate concentrations (6.4 microM), the clones isolated by sorting were already homogeneous at 0.8 microM methotrexate.

  13. Characterization of a recombinant humanized anti-cocaine monoclonal antibody produced from multiple clones for the selection of a master cell bank candidate.

    PubMed

    Wetzel, Hanna N; Webster, Rose P; Saeed, Fatima O; Kirley, Terence L; Ball, William J; Norman, Andrew B

    2017-06-03

    We have generated a humanized anti-cocaine monoclonal antibody (mAb), which is at an advanced stage of pre-clinical development. We report here in vitro binding affinity studies, and in vivo pharmacokinetic and efficacy studies of the recombinant mAb. The overall aim was to characterize the recombinant antibody from each of the three highest producing transfected clones and to select one to establish a master cell bank. In mAb pharmacokinetic studies, after injection with h2E2 (120 mg/kg iv) blood was collected from the tail tip of mice over 28 days. Antibody concentrations were quantified using ELISA. The h2E2 concentration as a function of time was fit using a two-compartment pharmacokinetic model. To test in vivo efficacy, mice were injected with h2E2 (120 mg/kg iv), then one hour later injected with an equimolar dose of cocaine. Blood and brain were collected 5 min after cocaine administration. Cocaine concentrations were quantified using LC/MS. The affinity of the antibody for cocaine was determined using a [(3)H] cocaine binding assay. All three antibodies had long elimination half-lives, 2-5 nM Kd for cocaine, and prevented cocaine's entry into the brain by sequestering it in the plasma. Pharmacokinetic and radioligand binding assays supported designation of the highest producing clone 85 as the master cell bank candidate. Overall, the recombinant h2E2 showed favorable binding properties, pharmacokinetics, and in vivo efficacy. Copyright © 2017 Elsevier Inc. All rights reserved.

  14. Correction: BTI-Tnao38, a new cell line derived from Trichoplusia ni, is permissive for AcMNPV infection and produces high levels of recombinant proteins.

    PubMed

    Hashimoto, Yoshi; Zhang, Sheng; Zhang, Shiying; Chen, Yun-Ru; Blissard, Gary W

    2012-04-24

    After publication we discovered an error in the identification of the origin of the cell line reported in our article in BMC Biotechnology (2010, 10:50), entitled "Ao38, a new cell line from eggs of the black witch moth, Ascalapha odorata (Lepidoptera: Noctuidae), is permissive for AcMNPV infection and produces high levels of recombinant proteins". Upon analysis of primary A. odorata cultures, we found that they were contaminated with cells of Trichoplusia ni origin. The origin of the Ao38 cell line was determined as T. ni using three marker genes and the Ao38 cell line was renamed BTI-Tnao38. References to the origin of the cell line as Ascalapha odorata should be replaced with "a cell line of Trichoplusia ni origin". The absence of TNCL virus detection in the BTI-Tnao38 (Ao38) cell line was confirmed using a highly sensitive RT-PCR protocol capable of detecting TNCL virus RNA at approximately 0.018 copies/cell. Because of these observations, we have revised the title of the original article to "Correction: BTI-Tnao38, a new cell line derived from Trichoplusia ni, is permissive for AcMNPV infection and produces high levels of recombinant proteins" and two additional authors were added to reflect their contributions to the analysis of this cell line.

  15. Heterologous production and detection of recombinant directing 2-deoxystreptamine (DOS) in the non-aminoglycoside-producing host Streptomyces venezuelae YJ003.

    PubMed

    Kurumbang, Nagendra Prasad; Oh, Tae-Jin; Liou, Kwangkyoung; Sohng, Jae Kyung

    2008-05-01

    2-Deoxystreptamine is a core aglycon that is vital to backbone formation in various aminoglycosides. This core structure can be modified to develop hybrid types of aminoglycoside antibiotics. We obtained three genes responsible for 2-deoxystreptamine production, neo7, neo6, and neo5, which encode 2-deoxy-scyllo-inosose synthase, L-glutamine: 2-deoxy-scyllo-inosose aminotransferase, and dehydrogenase, respectively, from the neomycin gene cluster. These genes were cloned into pIBR25, a Streptomyces expression vector, resulting in pNDOS. The recombinant pNDOS was transformed into a non-aminoglycoside-producing host, Streptomyces venezuelae YJ003, for heterologous expression. Based on comparisons of the retention time on LC-ESI/MS and ESIMS data with those of the 2-deoxystreptamine standard, a compound produced by S. venezuelae YJ003/pNDOS was found to be 2-deoxystreptamine.

  16. Use of a Chimeric Hsp70 to Enhance the Quality of Recombinant Plasmodium falciparum S-Adenosylmethionine Decarboxylase Protein Produced in Escherichia coli

    PubMed Central

    Makhoba, Xolani Henry; Burger, Adélle; Coertzen, Dina; Zininga, Tawanda; Birkholtz, Lyn-Marie; Shonhai, Addmore

    2016-01-01

    S-adenosylmethionine decarboxylase (PfAdoMetDC) from Plasmodium falciparum is a prospective antimalarial drug target. The production of recombinant PfAdoMetDC for biochemical validation as a drug target is important. The production of PfAdoMetDC in Escherichia coli has been reported to result in unsatisfactory yields and poor quality product. The co-expression of recombinant proteins with molecular chaperones has been proposed as one way to improve the production of the former in E. coli. E. coli heat shock proteins DnaK, GroEL-GroES and DnaJ have previously been used to enhance production of some recombinant proteins. However, the outcomes were inconsistent. An Hsp70 chimeric protein, KPf, which is made up of the ATPase domain of E. coli DnaK and the substrate binding domain of P. falciparum Hsp70 (PfHsp70) has been previously shown to exhibit chaperone function when it was expressed in E. coli cells whose resident Hsp70 (DnaK) function was impaired. We proposed that because of its domain constitution, KPf would most likely be recognised by E. coli Hsp70 co-chaperones. Furthermore, because it possesses a substrate binding domain of plasmodial origin, KPf would be primed to recognise recombinant PfAdoMetDC expressed in E. coli. First, using site-directed mutagenesis, followed by complementation assays, we established that KPf with a mutation in the hydrophobic residue located in its substrate binding cavity was functionally compromised. We further co-expressed PfAdoMetDC with KPf, PfHsp70 and DnaK in E. coli cells either in the absence or presence of over-expressed GroEL-GroES chaperonin. The folded and functional status of the produced PfAdoMetDC was assessed using limited proteolysis and enzyme assays. PfAdoMetDC co-expressed with KPf and PfHsp70 exhibited improved activity compared to protein co-expressed with over-expressed DnaK. Our findings suggest that chimeric KPf may be an ideal Hsp70 co-expression partner for the production of recombinant plasmodial

  17. Purification and characterization of pre-S-containing hepatitis B surface antigens produced in recombinant mammalian cell culture.

    PubMed

    Youn, B W; Samanta, H

    1989-02-01

    Heterogeneous, pre-S-rich HBsAg particles were expressed in recombinant mammalian cell culture and purified to near homogeneity. The purification process comprises: concentration of cell culture medium, protein precipitation by poly(ethylene glycol), gel filtration column chromatography, isopycnic ultracentrifugation by KBr and sucrose density gradient ultracentrifugation. The resulting HBsAg product was greater than 98% pure, and contained much of pre-S1 and pre-S2 components. Scanning densitometry analysis of the silver-stained HBsAg product showed approximately 70-80% S protein, approximately 10-20% pre-S2 protein, and approximately 5-15% pre-S1 protein. It was estimated that the amount of HBV-specific DNA present the final product was less than 7 pg mg-1 HBsAg. Further biochemical analysis has demonstrated that the HBsAg particles are very heterogeneous in charge and density. Charge heterogeneity was quite random among the particles, but density heterogeneity could be related to the different amounts of pre-S2 component in the particles.

  18. Biochemical and immunological characterization of a recombinantly-produced antifungal cysteine proteinase inhibitor from green kiwifruit (Actinidia deliciosa).

    PubMed

    Popovic, Milica; Andjelkovic, Uros; Burazer, Lidija; Lindner, Buko; Petersen, Arnd; Gavrovic-Jankulovic, Marija

    2013-10-01

    Plant proteinase inhibitors are considered important defense molecules against insect and pathogen attack. The cysteine proteinase inhibitor (CPI) from green kiwifruit (Actinidia deliciosa) belongs to the cystatin family and shows potent antifungal activity (in vitro and in vivo). However, the low abundance of this molecule in fruit (6μg/g of fresh fruit) seems to limit further investigations on the interaction between phytocystatin and photopathogenic fungi. In this paper the cDNA of the kiwi CPI was expressed in Escherichia coli. Fifteen N-terminal amino acids were identified by Edman degradation, and 77% of the rCPI primary structure was confirmed by mass fingerprint. The structural homology of recombinant CPI (rCPI) to its natural counterpart has been clearly demonstrated in immunological assays (immunoblot and ELISA inhibition). Biological activity of rCPI was demonstrated in inhibition assay with cysteine proteinase papain (EC50 2.78nM). In addition, rCPI reveals antifungal properties toward pathogenic fungi (Alternaria radicina and Botrytis cinerea), which designates it as an interesting model protein for the exploration of plant phytocystatins - pathogen interactions. Understanding the molecular mechanisms of natural plant resistance could lead to the development of ecologically safe fungicides for controlling post-harvest diseases and maintaining food quality.

  19. A new pH indicator dye-based method for rapid and efficient screening of l-asparaginase producing microorganisms.

    PubMed

    Mihooliya, Kanti N; Nandal, Jitender; Swami, Laxmi; Verma, Himanshu; Chopra, Lipsy; Sahoo, Debendra K

    2017-12-01

    l-asparaginase is a pharmaceutically and industrially important enzyme as it has potential to treat different cancers and inhibit acrylamide formation in fried and baked food products. In the present study, an attempt was made to screen for new and novel l-asparaginase producers using a widely applied phenol red and bromothymol blue (BTB)(1) dye-based plate assay. Screening of four different soil samples for l-asparaginase producers resulted in the isolation of three new potential l-asparaginase producing bacteria. These three strains identified (by 16S rRNA sequencing) as a Pseudomonas resinovorans strain IGS-131, a Bacillus safensis strain IGS-81, and a Glutamicibacter arilaitensis strain ICS-13 with enzyme activities of 10.91 IU/ml, 6.65 IU/ml, and 1.47 IU/ml, respectively. These three strains of bacteria have not been reported as l-asparaginase producers previously. Also, we developed a new pH indicator dye-based plate assay for the screening of l-asparaginase producers after testing eight different pH indicator dyes. This cresol red dye-based method gave a better differentiable zone of hydrolysis and consistent results as compared to previously reported phenol red and BTB-based plate assay. It was also found to be efficient in comparison to all other dyes studied. It produced a bright yellow color at acidic pH (5.5) and turned into a dark red or maroon color when pH was increased (above 7.5). This finding is expected to make screening of all kinds of l-asparaginases more comfortable, rapid, and efficient. Copyright © 2017 Elsevier Inc. All rights reserved.

  20. T-lineage cells require the thymus, but not V(D)J recombination to produce IL-17A and regulate granulopoiesis in vivo

    PubMed Central

    Smith, Emily; von Vietinghoff, Sibylle; Stark, Matthew A.; Zarbock, Alexander; Sanders, John M.; Duley, Amanda; Rivera-Nieves, Jesus; Bender, Timothy P.; Ley, Klaus

    2010-01-01

    IL-17A and F regulate granulopoiesis and are produced by memory T cells. Recombinase activating gene (Rag)1−/− mice cannot produce mature T cells, but maintain normal neutrophil counts. Nude mice are neutropenic or have near-normal neutrophil counts, depending on the prevailing intestinal flora. Athymic nude mice can produce IL-17F, but not A. By contrast, thymi from Rag1−/− mice contain as much IL-17A as those from wild-type (WT) mice. IL-17A-producing cells are found in the double negative (DN)1 compartment of the Rag1−/− thymus, and express intracellular CD3. These cells colonize the spleen and MLN and secrete IL-17A in vitro following stimulation with IL-23, at a level similar to WT splenocytes. Adoptively transferred Rag1−/− or WT thymocytes correct neutrophil counts in neutropenic nude mice. We conclude that the development of IL-17A-producing T-lineage cells requires an intact thymic epithelium, but not V(D)J recombination. PMID:19843951

  1. Characterization of a plant-produced recombinant human secretory IgA with broad neutralizing activity against HIV

    PubMed Central

    Paul, Matthew; Reljic, Rajko; Klein, Katja; Drake, Pascal MW; van Dolleweerd, Craig; Pabst, Martin; Windwarder, Markus; Arcalis, Elsa; Stoger, Eva; Altmann, Friedrich; Cosgrove, Catherine; Bartolf, Angela; Baden, Susan; Ma, Julian K-C

    2014-01-01

    Recombinant Secretory IgA (SIgA) complexes have the potential to improve antibody-based passive immunotherapeutic approaches to combat many mucosal pathogens. In this report, we describe the expression, purification and characterization of a human SIgA format of the broadly neutralizing anti-HIV monoclonal antibody (mAb) 2G12, using both transgenic tobacco plants and transient expression in Nicotiana benthamiana as expression hosts (P2G12 SIgA). The resulting heterodecameric complexes accumulated in intracellular compartments in leaf tissue, including the vacuole. SIgA complexes could not be detected in the apoplast. Maximum yields of antibody were 15.2 μg/g leaf fresh mass (LFM) in transgenic tobacco and 25 μg/g LFM after transient expression, and assembly of SIgA complexes was superior in transgenic tobacco. Protein L purified antibody specifically bound HIV gp140 and neutralised tier 2 and tier 3 HIV isolates. Glycoanalysis revealed predominantly high mannose structures present on most N-glycosylation sites, with limited evidence for complex glycosylation or processing to paucimannosidic forms. O-glycan structures were not identified. Functionally, P2G12 SIgA, but not IgG, effectively aggregated HIV virions. Binding of P2G12 SIgA was observed to CD209 / DC-SIGN, but not to CD89 / FcalphaR on a monocyte cell line. Furthermore, P2G12 SIgA demonstrated enhanced stability in mucosal secretions in comparison to P2G12 IgG mAb. PMID:25484063

  2. Standardization of allergen products: 1. Detailed characterization of GMP-produced recombinant Bet v 1.0101 as biological reference preparation.

    PubMed

    Himly, M; Nony, E; Chabre, H; Van Overtvelt, L; Neubauer, A; van Ree, R; Buchheit, K-H; Vieths, S; Moingeon, P; Ferreira, F

    2009-07-01

    Standardization of allergen extracts requires the availability of well-characterized recombinant allergens, which can be used as reference standards provided by the European regulatory authorities. The objective of this study was the detailed physicochemical and immunological characterization of rBet v 1.0101, which shall be used in a ring trial within the framework of the Biological Standardization Programme BSP090 of the European Directorate for Quality of Medicines and Healthcare. Recombinant Bet v 1.0101 Y0487 was produced under good manufacturing practice conditions and analysed by an array of physicochemical and immunological methods for identity, quantity, homogeneity, folding and denaturation, aggregation state and stability in solution, as well as biological activity. Batch Y0487 was shown to contain monomeric and well-folded protein being identical with rBet v 1.0101, as determined by mass spectrometry. SDS-PAGE, isoelectric focusing, deamidation analysis and size-exclusion chromatography with light scattering revealed sample homogeneity of >99.9%. Upon storage at +4 degrees C batch Y0487 retained the monomeric state up to 3 months. Protein quantification determined by amino acid analysis was found coinciding with half-maximal inhibition of serum IgE in ELISA. Biological activity of batch Y0487 was shown to be comparable to natural Bet v 1 by IgG and IgE immunoblotting, as well as basophil and T-cell activation. Recombinant Bet v 1.0101 Y0487 was characterized extensively by physicochemical and immunological methods. It was shown highly stable, monomeric and immunologically equivalent to its natural counterpart. Thus, it represents an appropriate candidate reference standard for Bet v 1.

  3. Development, evaluation, and laboratory validation of immunoassays for the diagnosis of equine infectious anemia (EIA) using recombinant protein produced from a synthetic p26 gene of EIA virus.

    PubMed

    Singha, Harisankar; Goyal, Sachin K; Malik, Praveen; Khurana, Sandip K; Singh, Raj K

    2013-12-01

    Equine infectious anemia (EIA)-a retroviral disease caused by equine infectious anemia virus (EIAV)-is a chronic, debilitating disease of horses, mules, and donkeys. EIAV infection has been reported worldwide and is recognized as pathogen of significant economic importance to the horse industry. This disease falls under regulatory control program in many countries including India. Control of EIA is based on identification of inapparent carriers by detection of antibodies to EIAV in serologic tests and "Stamping Out" policy. The current internationally accepted test for diagnosis of EIA is the agar gel immune-diffusion test (AGID), which detects antibodies to the major gag gene (p26) product. The objective of this study was to develop recombinant p26 based in-house immunoassays [enzyme linked immunosorbent assays (ELISA), and AGID] for EIA diagnosis. The synthetic p26 gene of EIAV was expressed in Escherichia coli and diagnostic potential of recombinant p26 protein were evaluated in ELISA and AGID on 7,150 and 1,200 equine serum samples, respectively, and compared with commercial standard AGID kit. The relative sensitivity and specificity of the newly developed ELISA were 100 and 98.6 %, respectively. Whereas, relative sensitivity and specificity of the newly developed AGID were in complete agreement in respect to commercial AGID kit. Here, we have reported the validation of an ELISA and AGID on large number of equine serum samples using recombinant p26 protein produced from synthetic gene which does not require handling of pathogenic EIAV. Since the indigenously developed reagents would be economical than commercial diagnostic kit, the rp26 based-immunoassays could be adopted for the sero-diagnosis and control of EIA in India.

  4. Vi Capsular Polysaccharide Produced by Recombinant Salmonella enterica Serovar Paratyphi A Confers Immunoprotection against Infection by Salmonella enterica Serovar Typhi.

    PubMed

    Xiong, Kun; Zhu, Chunyue; Chen, Zhijin; Zheng, Chunping; Tan, Yong; Rao, Xiancai; Cong, Yanguang

    2017-01-01

    Enteric fever is predominantly caused by Salmonella enterica serovar Typhi and Salmonella enterica serovar Paratyphi A, and accounts for an annual global incidence of 26.9 millions. In recent years, the rate of S. Paratyphi A infection has progressively increased. Currently licensed vaccines for typhoid fever, live Ty21a vaccine, Vi subunit vaccine, and Vi-conjugate vaccine, confer inadequate cross immunoprotection against enteric fever caused by S. Paratyphi A. Therefore, development of bivalent vaccines against enteric fever is urgently required. The immunogenic Vi capsular polysaccharide is characteristically produced in S. Typhi, but it is absent in S. Paratyphi A. We propose that engineering synthesis of Vi in S. Paratyphi A live-attenuated vaccine may expand its protection range to cover S. Typhi. In this study, we cloned the viaB locus, which contains 10 genes responsible for Vi biosynthesis, and integrated into the chromosome of S. Paratyphi A CMCC 50093. Two virulence loci, htrA and phoPQ, were subsequently deleted to achieve a Vi-producing attenuated vaccine candidate. Our data showed that, despite more than 200 passages, the viaB locus was stably maintained in the chromosome of S. Paratyphi A and produced the Vi polysaccharide. Nasal immunization of the vaccine candidate stimulated high levels of Vi-specific and S. Paratyphi A-specific antibodies in mice sera as well as total sIgA in intestinal contents, and showed significant protection against wild-type challenge of S. Paratyphi A or S. Typhi. Our study show that the Vi-producing attenuated S. Paratyphi A is a promising bivalent vaccine candidate for the prevention of enteric fever.

  5. Vi Capsular Polysaccharide Produced by Recombinant Salmonella enterica Serovar Paratyphi A Confers Immunoprotection against Infection by Salmonella enterica Serovar Typhi

    PubMed Central

    Xiong, Kun; Zhu, Chunyue; Chen, Zhijin; Zheng, Chunping; Tan, Yong; Rao, Xiancai; Cong, Yanguang

    2017-01-01

    Enteric fever is predominantly caused by Salmonella enterica serovar Typhi and Salmonella enterica serovar Paratyphi A, and accounts for an annual global incidence of 26.9 millions. In recent years, the rate of S. Paratyphi A infection has progressively increased. Currently licensed vaccines for typhoid fever, live Ty21a vaccine, Vi subunit vaccine, and Vi-conjugate vaccine, confer inadequate cross immunoprotection against enteric fever caused by S. Paratyphi A. Therefore, development of bivalent vaccines against enteric fever is urgently required. The immunogenic Vi capsular polysaccharide is characteristically produced in S. Typhi, but it is absent in S. Paratyphi A. We propose that engineering synthesis of Vi in S. Paratyphi A live-attenuated vaccine may expand its protection range to cover S. Typhi. In this study, we cloned the viaB locus, which contains 10 genes responsible for Vi biosynthesis, and integrated into the chromosome of S. Paratyphi A CMCC 50093. Two virulence loci, htrA and phoPQ, were subsequently deleted to achieve a Vi-producing attenuated vaccine candidate. Our data showed that, despite more than 200 passages, the viaB locus was stably maintained in the chromosome of S. Paratyphi A and produced the Vi polysaccharide. Nasal immunization of the vaccine candidate stimulated high levels of Vi-specific and S. Paratyphi A-specific antibodies in mice sera as well as total sIgA in intestinal contents, and showed significant protection against wild-type challenge of S. Paratyphi A or S. Typhi. Our study show that the Vi-producing attenuated S. Paratyphi A is a promising bivalent vaccine candidate for the prevention of enteric fever. PMID:28484685

  6. One-step enzymatic hydrolysis of starch using a recombinant strain of Saccharomyces cerevisiae producing alpha-amylase, glucoamylase and pullulanase.

    PubMed

    Janse, B J; Pretorius, I S

    1995-03-01

    A recombinant strain of Saccharomyces cerevisiae was constructed that contained the genes encoding a bacterial alpha-amylase (AMY1), a yeast glucoamylase (STA2) and a bacterial pullulanase (pulA). The Bacillus amyloliquefaciens alpha-amylase and S. cerevisiae var. diastaticus glucoamylase genes were expressed in S. cerevisiae using their native promoters and the encoded enzymes secreted under direction of their native leader sequences. In contrast, the Klebsiella pneumoniae pullulanase gene was placed under the control of the yeast alcohol dehydrogenase gene promoter (ADC1P) and secreted using the yeast mating pheromone alpha-factor secretion signal (MF alpha 1S). Transcription termination of the pullulanase gene was effected by the yeast tryptophan synthase gene terminator (TRP5T), whereas termination of the glucoamylase and alpha-amylase genes was directed by their native terminators. Pullulanase (PUL1) produced by recombinant yeasts containing ADC1P MF alpha 1S pulA TRP5T (designated PUL1) was further characterized and compared to its bacterial counterpart (PulA). The different genes were introduced into S. cerevisiae in different combinations and the various amylolytic Saccharomyces transformants compared to Schwanniomyces occidentalis. Introduction of PUL1 into a S. cerevisiae strain containing both STA2 and AMY1, resulted in 99% assimilation of starch.

  7. Spontaneous hybrids between native and exotic Rubus in the Western United States produce offspring both by apomixis and by sexual recombination

    PubMed Central

    Clark, L V; Jasieniuk, M

    2012-01-01

    Facultative asexual reproduction is a trait commonly found in invasive species. With a combination of sexual and asexual reproductive modes, such species may adapt to new environments via sexual recombination during range expansion, while at the same time having the benefits of asexuality such as the maintenance of fitness effects that depend upon heterozygosity. In the Western United States, native species of Rubus (Rosaceae) reproduce sexually whereas exotic naturalized Rubus species reproduce by pseudogamous apomixis. We hypothesized that new asexual lineages of Rubus could arise from hybridization in this range. To detect hybridization between native and exotic Rubus, we genotyped 579 individuals collected across California, Oregon and Washington with eight nuclear microsatellites and two chloroplast markers. Principal Coordinate Analysis and Bayesian clustering revealed a limited amount of hybridization of the native R. ursinus with the exotic R. armeniacus and R. pensilvanicus, as well as cultivated varieties. Genetic distances between these hybrids and their offspring indicated that both R. ursinus × R. armeniacus and R. ursinus × R. pensilvanicus produced a mix of apomictic and sexual seeds, with sexual seeds being more viable. Although neither of these hybrid types is currently considered invasive, they model the early stages of evolution of new invasive lineages, given the potential for fixed heterosis and the generation of novel genotypes. The hybrids also retain the ability to increase their fitness via sexual recombination and natural selection. Mixed reproductive systems such as those described here may be an important step in the evolution of asexual invasive species. PMID:22850699

  8. Spontaneous hybrids between native and exotic Rubus in the Western United States produce offspring both by apomixis and by sexual recombination.

    PubMed

    Clark, L V; Jasieniuk, M

    2012-11-01

    Facultative asexual reproduction is a trait commonly found in invasive species. With a combination of sexual and asexual reproductive modes, such species may adapt to new environments via sexual recombination during range expansion, while at the same time having the benefits of asexuality such as the maintenance of fitness effects that depend upon heterozygosity. In the Western United States, native species of Rubus (Rosaceae) reproduce sexually whereas exotic naturalized Rubus species reproduce by pseudogamous apomixis. We hypothesized that new asexual lineages of Rubus could arise from hybridization in this range. To detect hybridization between native and exotic Rubus, we genotyped 579 individuals collected across California, Oregon and Washington with eight nuclear microsatellites and two chloroplast markers. Principal Coordinate Analysis and Bayesian clustering revealed a limited amount of hybridization of the native R. ursinus with the exotic R. armeniacus and R. pensilvanicus, as well as cultivated varieties. Genetic distances between these hybrids and their offspring indicated that both R. ursinus × R. armeniacus and R. ursinus × R. pensilvanicus produced a mix of apomictic and sexual seeds, with sexual seeds being more viable. Although neither of these hybrid types is currently considered invasive, they model the early stages of evolution of new invasive lineages, given the potential for fixed heterosis and the generation of novel genotypes. The hybrids also retain the ability to increase their fitness via sexual recombination and natural selection. Mixed reproductive systems such as those described here may be an important step in the evolution of asexual invasive species.

  9. Productivity and quality of recombinant proteins produced by stable CHO cell clones can be predicted by transient expression in HEK cells.

    PubMed

    Diepenbruck, Carolin; Klinger, Matthias; Urbig, Thomas; Baeuerle, Patrick; Neef, Rüdiger

    2013-06-01

    Selection of lead candidates in drug discovery is a complex and time-consuming process. Here, we describe an approach that allows prediction of the productivity and quality of recombinant proteins by stable producer cell clones with the help of transient transfection studies. This is exemplified for three distinct bispecific T cell engager (BiTE(®))-a new class of single-chain antibody-based therapeutics showing very promising results in the treatment of cancer. BiTE(®) titers of transiently transfected HEK cells showed a striking correlation with titers of selected stable CHO cell clones. Likewise, the percentage of the monomeric BiTE(®) fraction in cell culture supernatants correlated well between transiently expressing HEK and stably expressing CHO cell clones. This validates the use of transient transfection studies for the selection of biopharmaceutical lead candidates with desired pharmaceutical properties.

  10. Expression, purification and characterization of yeast protein disulfide isomerase produced by a recombinant baculovirus-mediated silkworm, Bombyx mori, pupae expression system.

    PubMed

    Wang, Liyun; Shimizu, Yuri; Mizunaga, Takemitsu; Matsumoto, Shogo; Otsuka, Yuzuru

    2008-04-01

    Protein disulfide isomerase (PDI) is a multifunctional polypeptide presents in the endoplasmic reticulum of the cell. Silkworm (Bombyx mori) pupae were used as hosts to produce recombinant PDI (rPDI). The concentration-dependent chaperone activity of rPDI was evidenced by the inhibition of the aggregation of rhodanese. Approximately 297 microg rPDI was purified from a single silkworm pupa. Results of rPDI treated with endoglycosidase H and N-glycanase, PNGase F, indicate that non-N-glycosylated rPDI (occupying 90%) and N-glycosylated rPDI are expressed in the silkworm expression system. The difference in glycosylation between silkworm pupae and yeast is discussed.

  11. Recombinant HA1 produced in E. coli forms functional oligomers and generates strain-specific SRID potency antibodies for pandemic influenza vaccines

    PubMed Central

    Khurana, Surender; Larkin, Christopher; Verma, Swati; Joshi, Manju B.; Fontana, Juan; Steven, Alasdair C.; King, Lisa R.; Manischewitz, Jody; McCormick, William; Gupta, Rajesh K.; Golding, Hana

    2011-01-01

    Vaccine production and initiation of mass vaccination is a key factor in rapid response to new influenza pandemic. During the 2009–2010 H1N1 pandemic, several bottlenecks were identified, including the delayed availability of vaccine potency reagents. Currently, antisera for the single-radial immunodiffusion (SRID) potency assay are generated in sheep immunized repeatedly with HA released and purified after bromelain-treatment of influenza virus grown in eggs. This approach was a major bottleneck for pandemic H1N1 (H1N1pdm09) potency reagent development in 2009. Alternative approaches are needed to make HA immunogens for generation of SRID reagents in the shortest possible time. In this study, we found that properly folded recombinant HA1 globular domain (rHA1) from several type A viruses including H1N1pdm09 and two H5N1 viruses could be produced efficiently by using a bacterial expression system and subsequent purification. The rHA1 proteins were shown to form functional oligomers of trimers, similar to virus derived HA, and elicited high titer of neutralizing antibodies in rabbits and sheep. Importantly, the immune sera formed precipitation rings with reference antigens in the SRID assay in a dose-dependent manner. The HA contents in multiple H1N1 vaccine products from different manufacturers (and in several lots) as determined with the rHA1-generated sheep sera were similar to the values obtained with a traditionally generated sheep serum from NIBSC. We conclude that bacterially-expressed recombinant HA1 proteins can be produced rapidly and used to generate SRID potency reagents shortly after new influenza strains with pandemic potential are identified. PMID:21704111

  12. Recombinant lectins: an array of tailor-made glycan-interaction biosynthetic tools.

    PubMed

    Oliveira, Carla; Teixeira, José A; Domingues, Lucília

    2013-03-01

    Lectins are a heterogeneous group of proteins found in plants, animals and microorganisms, which possess at least one non-catalytic domain that binds reversibly to specific mono- or oligosaccharides. The range of lectins and respective biological activities is unsurprising given the immense diversity and complexity of glycan structures and the multiple modes of interaction with proteins. Recombinant DNA technology has been traditionally used for cloning and characterizing newly discovered lectins. It has also been employed as a means of producing pure and sequence-defined lectins for different biotechnological applications. This review focuses on the production of recombinant lectins in heterologous organisms, and highlighting the Escherichia coli and Pichia pastoris expression systems, which are the most employed. The choice of expression host depends on the lectin. Non-glycosylated recombinant lectins are produced in E. coli and post-translational modified recombinant lectins are produced in eukaryotic organisms, namely P. pastoris and non-microbial hosts such as mammalian cells. Emphasis is given to the applications of the recombinant lectins especially (a) in cancer diagnosis and/or therapeutics, (b) as anti-microbial, anti-viral, and anti-insect molecules or (c) in microarrays for glycome profiling. Most reported applications are from recombinant plant lectins. These applications benefit from the tailor-made design associated with recombinant production and will aid in unraveling the complex biological mechanisms of glycan-interactions, bringing recombinant lectins to the forefront of glycobiology. In conclusion, recombinant lectins are developing into valuable biosynthetic tools for biomedical research.

  13. The future of starch bioengineering: GM microorganisms or GM plants?

    PubMed

    Hebelstrup, Kim H; Sagnelli, Domenico; Blennow, Andreas

    2015-01-01

    Plant starches regularly require extensive modification to permit subsequent applications. Such processing is usually done by the use of chemical and/or physical treatments. The use of recombinant enzymes produced by large-scale fermentation of GM microorganisms is increasingly used in starch processing and modification, sometimes as an alternative to chemical or physical treatments. However, as a means to impart the modifications as early as possible in the starch production chain, similar recombinant enzymes may also be expressed in planta in the developing starch storage organ such as in roots, tubers and cereal grains to provide a GM crop as an alternative to the use of enzymes from GM microorganisms. We here discuss these techniques in relation to important structural features and modifications of starches such as: starch phosphorylation, starch hydrolysis, chain transfer/branching and novel concepts of hybrid starch-based polysaccharides. In planta starch bioengineering is generally challenged by yield penalties and inefficient production of the desired product. However, in some situations, GM crops for starch bioengineering without deleterious effects have been achieved.

  14. [Modern Approaches to the Creation of Industrial Microorganism Strains].

    PubMed

    Debabov, V G

    2015-04-01

    Microorganism producer strains are the basis of industrial biotechnology. Their properties determine the economical parameters of the production. Methods of rational design (metabolic engineering) and combinatorial methods of mutagenesis and selection (laboratory evolution, adaptive evolution, protein and genomic shuffling) are used for the construction of microorganism strains. Combination of these methods is frequently used. Modern strains usually do not contain plasmids and markers of drug resistance. All changes are introduced into the chromosome by the methods of homologous and site-specific recombination. The sum of such approaches is called recombineering. Gene expression is carried out at the optimal level under the control of promoters of a certain power (frequently regulated). Knowledge of a complete genomic sequence is almost a mandatory condition for the use of methods of metabolic engineering. Bioinformatics significantly assists in the selection of enzymes and the search for necessary genes and metabolic reactions. Measurement of metabolic fluxes largely assists in the construction of strains. The current level of science makes it possible to construct metabolic pathways de novo in strains for the production of chemicals and biofuel. Carbon dioxide has potential as a raw material for microbiological industry; therefore, the study of CO2 fixation by acetogens and electrogens is a promising direction of studies.

  15. Construction of a Recombinant Leuconostoc mesenteroides CJNU 0147 Producing 1,4-Dihydroxy-2-Naphthoic Acid, a Bifidogenic Growth Factor

    PubMed Central

    2015-01-01

    1,4-Dihydroxy-2-naphthoic acid (DHNA), a precursor of menaquinone (vitamin K2), has an effect on growth stimulation of bifidobacteria and prevention of osteoporosis, making it a promising functional food material. Therefore, we tried to clone the menB gene encoding DHNA synthase from Leuconostoc mesenteroides CJNU 0147. Based on the genome sequence of Leu. mesenteroides ATCC 8293 (GenBank accession no., CP000414), a primer set (Leu_menBfull_F and Leu_menBfull_R) was designed for the PCR amplification of menB gene of CJNU 0147. A DNA fragment (1,190 bp), including the menB gene, was amplified, cloned into pGEM-T Easy vector, and sequenced. The deduced amino acid sequence of MenB (DHNA synthase) protein of CJNU 0147 had a 98% similarity to the corresponding protein of ATCC 8293. The menB gene was subcloned into pCW4, a lactic acid bacteria - E. coli shuttle vector, and transferred to CJNU 0147. The transcription of menB gene of CJNU 0147 (pCW4::menB) was increased, when compared with those of CJNU 0147 (pCW4) and CJNU 0147 (−). The DHNA was produced from it at a detectable level, indicating that the cloned menB gene of CJNU 0147 encoded a DHNA synthase which is responsible for the production of DHNA, resulting in an increase of bifidogenic growth stimulation activity. PMID:26877648

  16. Construction of a Recombinant Leuconostoc mesenteroides CJNU 0147 Producing 1,4-Dihydroxy-2-Naphthoic Acid, a Bifidogenic Growth Factor.

    PubMed

    Eom, Ji-Eun; Moon, Gi-Seong

    2015-01-01

    1,4-Dihydroxy-2-naphthoic acid (DHNA), a precursor of menaquinone (vitamin K2), has an effect on growth stimulation of bifidobacteria and prevention of osteoporosis, making it a promising functional food material. Therefore, we tried to clone the menB gene encoding DHNA synthase from Leuconostoc mesenteroides CJNU 0147. Based on the genome sequence of Leu. mesenteroides ATCC 8293 (GenBank accession no., CP000414), a primer set (Leu_menBfull_F and Leu_menBfull_R) was designed for the PCR amplification of menB gene of CJNU 0147. A DNA fragment (1,190 bp), including the menB gene, was amplified, cloned into pGEM-T Easy vector, and sequenced. The deduced amino acid sequence of MenB (DHNA synthase) protein of CJNU 0147 had a 98% similarity to the corresponding protein of ATCC 8293. The menB gene was subcloned into pCW4, a lactic acid bacteria - E. coli shuttle vector, and transferred to CJNU 0147. The transcription of menB gene of CJNU 0147 (pCW4::menB) was increased, when compared with those of CJNU 0147 (pCW4) and CJNU 0147 (-). The DHNA was produced from it at a detectable level, indicating that the cloned menB gene of CJNU 0147 encoded a DHNA synthase which is responsible for the production of DHNA, resulting in an increase of bifidogenic growth stimulation activity.

  17. Microorganisms detected by enzyme-catalyzed reaction

    NASA Technical Reports Server (NTRS)

    Vango, S. P.; Weetall, H. H.; Weliky, N.

    1966-01-01

    Enzymes detect the presence of microorganisms in soils. The enzyme lysozymi is used to release the enzyme catalase from the microorganisms in a soil sample. The catalase catalyzes the decomposition of added hydrogen peroxide to produce oxygen which is detected manometrically. The partial pressure of the oxygen serves as an index of the samples bacteria content.

  18. Polyhydroxybutyrate: plastic made and degraded by microorganisms.

    PubMed

    Hankermeyer, C R; Tjeerdema, R S

    1999-01-01

    Polyhydroxybutyrate (PHB) offers many advantages over traditional petrochemically derived plastics. In addition to its complete biodegradability, PHB is formed from renewable resources. It possesses better physical properties than polypropylene for food packaging applications and is completely nontoxic. The poor low-impact strength of PHB is solved by incorporation of hydroxyvalerate monomers into the polymer to produce polyhydroxybutyrate-co-valerate (PHBV), which is commercially marketed under the trade name Biopol. Like PHB, PHBV completely degrades into carbon dioxide and water under aerobic conditions. Microbial synthesis of PHB is the best method for industrial production because it ensures the proper stereochemistry for biodegradation. Microorganisms synthesize and store PHB under nutrient-limited conditions and degrade and metabolize it when the limitation is removed. Current production employs Alcaligenes eutrophus because it grows efficiently on glucose as a carbon source, accumulates PHB up to 80% of its dry weight, and is able to synthesize PHBV when propionic acid is added to the feedstock. PHBV is currently 16 times the price of polypropylene. However, the development of transgenic PHA-producing organisms is expected to greatly reduce its cost. Benefits of using transgenic systems include lack of a depolymerase system, ability to use faster-growing organisms, production of highly purified polymers, and ability to utilize inexpensive carbon sources. Because transgenic plants may someday result in the evolution of plastic crops that could lower the price of PHA to a competitive level, future research will surely focus on such recombinant DNA techniques.

  19. Beneficial microorganisms [Chapter 14

    Treesearch

    Kim M. Wilkinson

    2009-01-01

    The web of life depends on microorganisms, a vast network of small and unseen allies that permeate the soil, water, and air of our planet. For people who work with plants, the greatest interest in microorganisms is in the complex communities that are part of the soil. Beneficial microorganisms are naturally occurring bacteria, fungi, and other microbes that play a...

  20. Polysaccharides from Extremophilic Microorganisms

    NASA Astrophysics Data System (ADS)

    Nicolaus, B.; Moriello, V. Schiano; Lama, L.; Poli, A.; Gambacorta, A.

    2004-02-01

    Several marine thermophilic strains were analyzed for exopolysaccharide production. The screening process revealed that a significant number of thermophilic microorganisms were able to produce biopolymers, and some of them also revealed interesting chemical compositions. We have identified four new polysaccharides from thermophilic marine bacteria, with complex primary structures and with different repetitive units: a galacto-mannane type from strain number 4004 and mannane type for the other strains. The thermophilic Bacillus thermantarcticus produces two exocellular polysaccharides (EPS 1, EPS 2) that give the colonies a typical mucous character. The exopolysaccharide fraction was produced with all substrates assayed, although a higher yield 400 mg liter-1 was obtained with mannose as carbon and energy source. NMR spectra confirmed that EPS 1 was a heteropolysaccharide of which the repeating unit was constituted by four different α-D-mannoses and three different β-D-glucoses. It seems to be close to some xantan polymers. EPS 2 was a mannan. Four different α-D-mannoses were found as the repeating unit. Production and chemical studies of biopolymers produced by halophilic archaea, Haloarcula species were also reported.

  1. Recombinant gonadotropins.

    PubMed

    Lathi, R B; Milki, A A

    2001-10-01

    Recombinant DNA technology makes it possible to produce large amounts of human gene products for pharmacologic applications, supplanting the need for human tissues. The genes for the alpha and beta subunits of follicle-stimulating hormone (FSH), luteinizing hormone (LH), and human chorionic gonadotropin (hCG) have been characterized and cloned. Recombinant FSH (rFSH) has been shown to be safe and effective in the treatment of fertility disorders. In comparison with the urinary gonadotropin products, human menopausal gonadotropins (HMG), and urinary follitropins (uFSH), rFSH is more potent and better tolerated by patients. Recombinant HCG appears to be as efficacious as urinary HCG with the benefit of improved local tolerance. Recombinant LH (rLH) is likely to be recommended as a supplement to rFSH for ovulation induction in hypogonadotropic women. It may also benefit in vitro fertilization patients undergoing controlled ovarian hyperstimulation with rFSH combined with pituitary suppression, with a gonadotropin-releasing hormone agonist or antagonist.

  2. Human anti-varicella-zoster virus (VZV) recombinant monoclonal antibody produced after Zostavax immunization recognizes the gH/gL complex and neutralizes VZV infection.

    PubMed

    Birlea, Marius; Owens, Gregory P; Eshleman, Emily M; Ritchie, Alanna; Traktinskiy, Igor; Bos, Nathan; Seitz, Scott; Azarkh, Yevgeniy; Mahalingam, Ravi; Gilden, Don; Cohrs, Randall J

    2013-01-01

    Varicella-zoster virus (VZV) is a ubiquitous, highly cell-associated, and exclusively human neurotropic alphaherpesvirus. VZV infection is initiated by membrane fusion, an event dependent in part on VZV glycoproteins gH and gL. Consistent with its location on the virus envelope, the gH/gL complex is a target of neutralizing antibodies produced after virus infection. One week after immunizing a 59-year-old VZV-seropositive man with Zostavax, we sorted his circulating blood plasma blasts and amplified expressed immunoglobulin variable domain sequences by single-cell PCR. Sequence analysis identified two plasma blast clones, one of which was used to construct a recombinant monoclonal antibody (rec-RC IgG). The rec-RC IgG colocalized with VZV gE on the membranes of VZV-infected cells and neutralized VZV infection in tissue culture. Mass spectrometric analysis of proteins immunoprecipitated by rec-RC IgG identified both VZV gH and gL. Transfection experiments showed that rec-RC IgG recognized a VZV gH/gL protein complex but not individual gH or gL proteins. Overall, our recombinant monoclonal anti-VZV antibody effectively neutralizes VZV and recognizes a conformational epitope within the VZV gH/L protein complex. An unlimited supply of this antibody provides the opportunity to analyze membrane fusion events that follow virus attachment and to identify multiple epitopes on VZV-specific proteins.

  3. Identification and characterization of a -1 reading frameshift in the heavy chain constant region of an IgG1 recombinant monoclonal antibody produced in CHO cells

    PubMed Central

    Lian, Zhirui; Wu, Qindong; Wang, Tongtong

    2016-01-01

    ABSTRACT Frameshifts lead to complete alteration of the intended amino acid sequences, and therefore may affect the biological activities of protein therapeutics and pose potential immunogenicity risks. We report here the identification and characterization of a novel -1 frameshift variant in a recombinant IgG1 therapeutic monoclonal antibody (mAb) produced in Chinese hamster ovary cells during the cell line selection studies. The variant was initially observed as an atypical post-monomer fragment peak in size exclusion chromatography. Characterization of the fragment peak using intact and reduced liquid chromatography-mass spectrometry (LC-MS) analyses determined that the fragment consisted of a normal light chain disulfide-linked to an aberrant 26 kDa fragment that could not be assigned to any HC fragment even after considering common modifications. Further analysis using LC-MS/MS peptide mapping revealed that the aberrant fragment contained the expected HC amino acid sequence (1-232) followed by a 20-mer novel sequence corresponding to expression of heavy chain DNA sequence in the -1 reading frame. Examination of the DNA sequence around the frameshift initiation site revealed that a mononucleotide repeat GGGGGG located in the IgG1 HC constant region was most likely the structural root cause of the frameshift. Rapid identification of the frameshift allowed us to avoid use of a problematic cell line containing the frameshift as the production cell line. The frameshift reported here may be observed in other mAb products and the hypothesis-driven analytical approaches employed here may be valuable for rapid identification and characterization of frameshift variants in other recombinant proteins. PMID:26652198

  4. Effector properties and glycosylation patterns of recombinant human anti-D-IgG1 antibodies produced by human PER.C6(®) cells.

    PubMed

    Olovnikova, N I; Grigorieva, O V; Petrov, A V

    2012-12-01

    Creation of effective monoclonal anti-D immunoglobulin for prevention of hemolytic disease of the newborn remains an unsolved problem because there is still no producer cell strain providing stable production and adequate glycosylation of antibodies. Recombinant anti-D have been obtained on the basis of human PER.C6(®) cells and characterized. Anti-D antibodies expressed in PER.C6(®) exhibited lower hemolytic activity in antibody-dependent cytotoxicity (ADCC) reaction mediated by low-affinity Fcγ receptors in comparison with identical antibodies of lymphoblastoid origin. Monoclonal antibodies produced by PER.C6(®) are completely fucosylated and desialylated, i.e. are characterized by abnormal glycosylation. Addition of kifunensine (α-mannosidase I inhibitor) to the medium led to production of antibodies with high hemolytic activity. Reduced activity of monoclonal antibodies in PER.C6(®) cells and the effect of kifunensine (causing synthesis of defucosylated glycans) suggest that the absence of fucose is the key factor responsible for Fc affinity for low-affinity receptors.

  5. [Spreading and mechanisms of antimicrobial resistance of microorganisms, producing beta-lactamases. Phenotypical screening for MBL producers (carbapenemases B1) among strains of Pseudomonas genus, isolated in cases of nosocomial infections].

    PubMed

    Ivanov, D V; Egorov, A M

    2007-01-01

    Intrahospital strains (215) of the bacterial genus Pseudomonas isolated from patients of 30 Medical centers of 15 Russian regions have been investigated for antibiotic resistance. The bacterial cultures resistant to imipenem and/or meropenem were considered as metallo-beta-lactamase (MBL) producers. Production of subclass B1 MBL (carbapenemases) was evaluated by means of the double-disk approximation test using MBL inhibitor, EDTA. There were 55 P. aeroginosa strains (25.6%) resistant to imipenem and meropenem simultaneously; 19 isolates (8.8%) of P. aeroginosa were characterized by synergism between carbapenem and EDTA. The subclass B1 MBL producers are widely distributed in the intrahospital strain obtained from Moscow, Yaroslavl, Ekaterinburg, Omsk, and Tomsk hospitals.

  6. Microorganism Utilization for Synthetic Milk

    NASA Technical Reports Server (NTRS)

    Morford, Megan A.; Khodadad, Christina L.; Caro, Janicce I.; Spencer, LaShelle E.; Richards, Jeffery T.; Strayer, Richard F.; Birmele, Michele N.; Wheeler, Raymond M.

    2014-01-01

    A desired architecture for long duration spaceflight, like aboard the International Space Station or for future missions to Mars, is to provide a supply of fresh food crops for the astronauts. However, some crops can create a high proportion of inedible plant waste. The main goal of the Synthetic Biology project, Cow in a Column, was to produce the components of milk (sugar, lipid, protein) from inedible plant waste by utilizing microorganisms (fungi, yeast, bacteria). Of particular interest was utilizing the valuable polysaccharide, cellulose, found in plant waste, to naturally fuel-through microorganism cellular metabolism- the creation of sugar (glucose), lipid (milk fat), and protein (casein) in order to produce a synthetic edible food product. Environmental conditions such as pH, temperature, carbon source, aeration, and choice microorganisms were optimized in the laboratory and the desired end-products, sugars and lipids, were analyzed. Trichoderma reesei, a known cellulolytic fungus, was utilized to drive the production of glucose, with the intent that the produced glucose would serve as the carbon source for milk fat production and be a substitute for the milk sugar lactose. Lipid production would be carried out by Rhodosporidium toruloides, yeast known to accumulate those lipids that are typically found in milk fat. Results showed that glucose and total lipid content were below what was expected during this phase of experimentation. In addition, individual analysis of six fatty acids revealed that the percentage of each fatty acid was lower than naturally produced bovine milk. Overall, this research indicates that microorganisms could be utilized to breakdown inedible solid waste to produce useable products. For future work, the production of the casein protein for milk would require the development of a genetically modified organism, which was beyond the scope of the original project. Additional trials would be needed to further refine the required

  7. Scintillation proximity assay for human DNA topoisomerase I using recombinant biotinyl-fusion protein produced in baculovirus-infected insect cells.

    PubMed

    Lerner, C G; Saiki, A Y

    1996-09-05

    DNA topoisomerases are well-established targets of important therapeutic agents which include the antibacterial quinolones and anticancer camptothecins. Screens for new classes of topoisomerase inhibitors generally employ methods, such as gel electrophoresis, which are not readily amenable to a rapid high-throughput format. We describe here a high-throughput assay to screen for inhibitors of human DNA topoisomerase I based on the scintillation proximity assay. The assay employs recombinant biotinyl-topoisomerase I fusion protein, a hybrid protein which contains a domain that is biotinylated during in vivo expression. The hybrid topoisomerase I fusion protein is found to be biotinylated, active, and nuclear-localized when produced in insect cells using a baculovirus expression system. The biotinyl-topoisomerase I fusion protein can be captured from crude nuclear extracts by immobilization on streptavidin-coated scintillation proximity assay beads. The assay detects binding of 3H-labeled DNA to the bead-immobilized enzyme by scintillation counting. The method is also able to detect stabilization of covalent protein-DNA complexes by camptothecin, an inhibitor previously shown to stabilize covalent intermediates that form during catalysis.

  8. IL-10-IFN-γ Double Producers CD4+ T Cells Are Induced by Immunization with an Amastigote Stage Specific Derived Recombinant Protein of Trypanosoma Cruzi

    PubMed Central

    Flores-García, Yevel; Rosales-Encina, José Luis; Satoskar, Abhay R.; Talamás-Rohana, Patricia

    2011-01-01

    During the acute phase of infection, T. cruzi replicates extensively and releases immunomodulatory molecules that delay parasite-specific responses mediated by effector T cells. This mechanism of evasion allows the parasite to spread in the host. Parasite molecules that regulate the host immune response during Chagas'disease have not been fully identified. GPI-anchored mucins, glycoinositolphospholipids, and glycoproteins comprise some of the most abundant T. cruzi surface molecules. IL-10 IFN-γ-secreting CD4+ T cells are activated during chronic infections and are responsible for prolonged persistence of parasite and for host protection against severe inflammatory responses. In this work we evaluated the role of rMBP::SSP4 protein of T. cruzi, a recombinant protein derived from a GPI anchored antigen, SSP4, as an immunomodulator molecule, finding that it was able to induce high concentrations of IL-10 and IFN-γ both in vivo and in vitro; during this last condition, both cytokines were produced by IL-10-IFN-γ-secreting CD4+ T cells. PMID:21927578

  9. Bactericidal properties of the antimicrobial peptide Ib-AMP4 from Impatiens balsamina produced as a recombinant fusion-protein in Escherichia coli.

    PubMed

    Fan, Xiaobo; Schäfer, Holger; Reichling, Jürgen; Wink, Michael

    2013-10-01

    Antimicrobial peptides (AMPs) represent a novel class of powerful natural antimicrobial agents. As AMPs are bactericidal, production of AMPs in recombinant bacteria is far from trivial. We report the production of Impatiens balsamina antimicrobial peptide 4 (Ib-AMP4, originally isolated from Impatiens balsamina) in Escherichia coli as a fusion protein and investigate Ib-AMP4's antimicrobial effects on human pathogens. A plasmid vector pET32a-Trx-Ib-AMP4 was constructed and transferred into E. coli. After induction, a soluble fusion protein was expressed successfully. The Ib-AMP4 peptide was obtained with a purity of over 90% after nickel affinity chromatography, ultrafiltration, enterokinase cleavage and sephadex size exclusion chromatography. For maximum activity, Ib-AMP4, which possesses two disulfide bonds, required activation with 5 μg/mL H2 O2 . Antimicrobial assays showed that Ib-AMP4 could efficiently target clinical multiresistant isolates including methicillin-resistant Staphylococcus aureus and extended-spectrum β-lactamase-producing E. coli. Time kill experiments revealed that Ib-AMP4 is bactericidal within 10 min after application. Haemolysis and cytotoxicity assays implied selectivity towards bacteria, an important prerequisite for clinical applications. Ib-AMP4 might be an interesting candidate for clinical studies involving patients with septicemia or for coating clinical devices, such as catheters. The method described here may be applicable for expression and purification of other AMPs with multiple disulfide bridges.

  10. The quest for industrial enzymes from microorganisms.

    PubMed

    Yamaguchi, Shotaro

    2017-01-01

    Satoshi Ōmura, Professor Emeritus at Kitasato University, was awarded the Nobel Prize for his discovery of a substance of tremendous value to mankind from a microorganism. As a researcher who regularly deals with enzymes produced by microorganisms and a person engaged in microorganism-based business, Professor Ōmura's Nobel Prize fills me with great pride and joy. It is perhaps not surprising that this Nobel Prize-winning research would emerge from Asia, specifically Japan, where people live in harmony with nature rather than try to conquer it. At Amano Enzyme Inc., we devote ourselves to searching for novel enzymes from microorganisms. While incorporating my own experiences, I will recount the stories of a few discoveries of valuable enzyme-producing microbes in soil and bacterial strain libraries. I will also briefly introduce microbial strain library construction as a tool for facilitating the identification of the desired producing bacteria.

  11. Engineering Microorganisms for Energy Production

    DTIC Science & Technology

    2006-06-01

    photovoltaic solar cells). For engineered microorganisms to succeed in the marketplace , their systems costs need to be significantly lower; however we are not...hydrogenase enzymes to produce molecular hydrogen. This report will assess the fundamental requirements and the technical bar- riers that need to be overcome...and photosynthetic engineering from the standpoint of individual components (e.g., designing better enzymes for catalyzing critical reactions), the

  12. Recombinant organisms capable of fermenting cellobiose

    DOEpatents

    Ingram, Lonnie O.; Lai, Xiaokuang; Moniruzzaman, Mohammed; York, Sean W.

    2000-01-01

    This invention relates to a recombinant microorganism which expresses pyruvate decarboxylase, alcohol dehydrogenase, Klebsiella phospho-.beta.-glucosidase and Klebsiella (phosphoenolpyruvate-dependent phosphotransferase system) cellobiose-utilizing Enzyme II, wherein said phospho-.beta.-glucosidase and said (phosphoenolpyruvate-dependent phosphotransferase) cellobiose-utilizing Enzyme II are heterologous to said microorganism and wherein said microorganism is capable of utilizing both hemicellulose and cellulose, including cellobiose, in the production of ethanol.

  13. Microorganisms and Chemical Pollution

    ERIC Educational Resources Information Center

    Alexander, M.

    1973-01-01

    Discusses the importance of microorganisms in chemical pollution and pollution abatement. Selected chemical pollutants are chosen to illustrate that microorganisms synthesize hazardous substances from reasonably innocuous precursors, while others act as excellent environmental decontaminating agents by removing undesirable natural and synthetic…

  14. Microorganisms and Chemical Pollution

    ERIC Educational Resources Information Center

    Alexander, M.

    1973-01-01

    Discusses the importance of microorganisms in chemical pollution and pollution abatement. Selected chemical pollutants are chosen to illustrate that microorganisms synthesize hazardous substances from reasonably innocuous precursors, while others act as excellent environmental decontaminating agents by removing undesirable natural and synthetic…

  15. Functionalized silk assembled from a recombinant spider silk fusion protein (Z-4RepCT) produced in the methylotrophic yeast Pichia pastoris.

    PubMed

    Jansson, Ronnie; Lau, Cheuk H; Ishida, Takuya; Ramström, Margareta; Sandgren, Mats; Hedhammar, My

    2016-05-01

    Functional biological materials are a growing research area with potential applicability in medicine and biotechnology. Using genetic engineering, the possibility to introduce additional functions into spider silk-based materials has been realized. Recently, a recombinant spider silk fusion protein, Z-4RepCT, was produced intracellularly in Escherichia coli and could after purification self-assemble into silk-like fibers with ability to bind antibodies via the IgG-binding Z domain. In this study, the use of the methylotrophic yeast Pichia pastoris for production of Z-4RepCT has been investigated. Temperature, pH and production time were influencing the amount of soluble Z-4RepCT retrieved from the extracellular fraction. Purification of secreted Z-4RepCT resulted in a mixture of full-length and degraded silk proteins that failed to self-assemble into fibers. A position in the C-terminal domain of 4RepCT was identified as being subjected to proteolytic cleavage by proteases in the Pichia culture supernatant. Moreover, the C-terminal domain was subjected to glycosylation during production in P. pastoris. These observed alterations of the CT domain are suggested to contribute to the failure in fiber assembly. As alternative approach, Z-4RepCT retrieved from the intracellular fraction, which was less degraded, was used and shown to retain ability to assemble into silk-like fibers after enzymatic deglycosylation. Copyright © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  16. Improvement in the stability and functionality of Nicotiana tabacum produced recombinant TRAIL through employment of endoplasmic reticulum expression and ascorbate buffer mediated extraction strategies

    PubMed Central

    Heidari, Hamid Reza; Bandehpour, Mojgan; Vahidi, Hossein; Barar, Jaleh; Kazemi, Bahram; Naderi-Manesh, Hossein

    2014-01-01

    Introduction: In order to employ Nicotiana tabacum cells as a profitable natural bioreactor for production of bio-functional "Soluble human TRAIL" (ShTRAIL), endoplasmic reticulum (ER) targeted expression and innovative extraction procedures were exploited. Methods: At first, the ShTRAIL encoding gene was sub-cloned into designed H2 helper vector to equip it with potent TMV omega leader sequences, ER sorting signal peptide, poly-histidine tag and ER retention signal peptide (KDEL). Then, the ER targeted ShTRAIL cassette was sequentially sub-cloned into "CaMV-35S" helper and "pGreen-0179" final expression vectors. Afterward, Agrobacterium mediated transformation method was adopted to express the ShTRAIL in the ER of N. tabacum . Next, the ShTRAIL protein was extracted through both phosphate and innovative ascorbate extraction buffers. Subsequently, oligomerization state of the ShTRAIL was evaluated through cross-linking assay and western blot analysis. Then, semi-quantitative western blot analysis was performed to estimate the ShTRAIL production. Finally, biological activity of the ShTRAIL was evaluated through MTT assay. Results: The phosphate buffer extracted ShTRAIL was produced in dimmer form, whereas the ShTRAIL extracted with ascorbate buffer generated trimer form. The ER targeted ShTRAIL strategy increased the ShTRAIL’s production level up to about 20 μg/g of fresh weight of N. tabacum . MTT assay indicated that ascorbate buffer extracted ShTRAIL could prohibit proliferation of A549 cell line. Conclusion: Endoplasmic reticulum expression and reductive ascorbate buffer extraction procedure can be employed to enhance the stability and overall production level of bio-functional recombinant ShTRAIL from transgenic N. tabacum cells. PMID:25337465

  17. On-line casein micelle disruption for downstream purification of recombinant human myelin basic protein produced in the milk of transgenic cows.

    PubMed

    Al-Ghobashy, Medhat A; Williams, Martin A K; Brophy, Brigid; Laible, Götz; Harding, David R K

    2009-06-01

    Downstream purification of a model recombinant protein (human myelin basic protein) from milk of transgenic cows is described. The recombinant protein was expressed as a His tagged fusion protein in the milk of transgenic cows and was found associated with the casein micellar phase. While difficulties in obtaining good recoveries were found when employing conventional micelle disruption procedures, direct capture using the cation exchanger SP Sepharose Big Beads was found successful in the extraction of the recombinant protein. Early breakthrough suggested a slow release of the recombinant protein from the micelles and dictated micelle disruption in order to obtain good yields. A new approach for deconstruction of the calcium core of the casein micelles, employing the interaction between the micellar calcium and the active sites of the cation exchanger resin was developed. Milk samples were loaded to the column in aliquots with a column washing step after each aliquot. This sequential loading approach successfully liberated the recombinant protein from the micelles and was found superior to the conventional sample loading approach. It increased the recovery by more than 25%, reduced fouling due to milk components and improved the column hydrodynamic properties as compared to the conventional sample loading approach. Hardware and software modifications to the chromatography system were necessary in order to keep the whole process automated. A second purification step using a Ni2+ affinity column was used to isolate the recombinant protein at purity more than 90% and a recovery percentage of 78%.

  18. Granulocyte/macrophage-colony stimulating factor produced by recombinant avian poxviruses enriches the regional lymph nodes with antigen-presenting cells and acts as an immunoadjuvant.

    PubMed

    Kass, E; Panicali, D L; Mazzara, G; Schlom, J; Greiner, J W

    2001-01-01

    Recombinant avian poxviruses [fowlpox and canarypox (ALVAC)], restricted for replication in nonavian cell substrates and expressing granulocyte/macrophage-colony stimulating factor (avipox-GM-CSF), were evaluated for their ability to enrich an immunization site with antigen-presenting cells (APCs) and, in turn, function as biological vaccine adjuvants. Avipox-GM-CSF administered as a single s.c. injection significantly enhanced the percentage and absolute number of APCs in the regional lymph nodes that drain the injection site. Both the magnitude and duration of the cellular and phenotypic increases within the lymph nodes induced by the avipox-GM-CSF viruses were significantly (P < 0.05) greater than those measured in mice treated with four daily injections of recombinant GM-CSF protein. Temporal studies revealed that the APC enrichment of regional lymph nodes was sustained for 21-28 days after injection of the recombinant avipox virus expressing GM-CSF and, moreover, three injections of the recombinant virus could be given without any appreciable loss of in vivo bioactivity. Mice expressing human carcinoembryonic antigen (CEA) as a transgene (CEA.Tg) developed CEA-specific humoral and cell-mediated immunity after being immunized with avipox-CEA. The coadministration of recombinant avipox viruses expressing CEA and GM-CSF significantly enhanced CEA-specific host immunity with an accompanying immunotherapeutic response in tumor-bearing CEA.Tg mice. The optimal use of avipox-GM-CSF, in terms of dose and dose schedule, especially when used with different immunogens, remains to be determined. Nonetheless, the present findings demonstrate: (a) the effective delivery of GM-CSF to an immunization site using a recombinant avian poxvirus; (b) the compatibility of delivering an antigen and GM-CSF in replication-defective viruses to enhance antigen-specific immunity; and (c) the combined use of recombinant avipox viruses expressing CEA and GM-CSF to generate antitumor

  19. Enhanced production of carboxymethylcellulase of a marine microorganism, Bacillus subtilis subsp. subtilis A-53 in a pilot-scaled bioreactor by a recombinant Escherichia coli JM109/A-53 from rice bran.

    PubMed

    Lee, Eun-Jung; Lee, Bo-Hwa; Kim, Bo-Kyung; Lee, Jin-Woo

    2013-05-01

    A gene encoding the carboxymethylcellulase (CMCase) of a marine bacterium, Bacillus subtilis subsp. subtilis A-53, was cloned in Escherichia coli JMB109 and the recombinant strain was named as E. coli JMB109/A-53. The optimal conditions of rice bran, ammonium chloride, and initial pH of the medium for cell growth, extracted by Design Expert Software based on response surface methodology, were 100.0 g/l, 7.5 g/l, and 7.0, respectively, whereas those for production of CMCase were 100.0 g/l, 7.5 g/l, and 8.0. The optimal temperatures for cell growth and the production of CMCase by E. coli JM109/A-53 were found to be and 40 and 35 °C, respectively. The optimal agitation speed and aeration rate of a 7 l bioreactor for cell growth were 400 rpm and 1.5 vvm, whereas those for production of CMCase were 400 rpm and 0.5 vvm. The optimal inner pressure for cell growth was 0.06 MPa, which was the same as that for production of CMCase. The production of CMCase by E. coli JM109/A-53 under optimized conditions was 880.2 U/ml, which was 2.9 times higher than that before optimization. In this study, rice bran and ammonium chloride were developed as carbon and nitrogen source for production of CMCase by a recombinant E. coli JM109/A-53 and the productivity of E. coli JM109/A-53 was 5.9 times higher than that of B. subtilis subp. subtilis A-53.

  20. Anti-Bacterial Activity of Recombinant Human β-Defensin-3 Secreted in the Milk of Transgenic Goats Produced by Somatic Cell Nuclear Transfer

    PubMed Central

    Han, Chengquan; Zhang, Hui; Wang, Yongsheng; Su, Jianmin; Quan, Fusheng; Gao, Mingqing; Zhang, Yong

    2013-01-01

    The present study was conducted to determine whether recombinant human β-defensin-3 (rHBD3) in the milk of transgenic goats has an anti-bacterial activity against Escherichia coli (E. coli), Staphylococcus aureus (S. aureus) and Streptococcus agalactiae (S. agalactiae) that could cause mastitis. A HBD3 mammary-specific expression vector was transfected by electroporation into goat fetal fibroblasts which were used to produce fourteen healthy transgenic goats by somatic cell nuclear transfer. The expression level of rHBD3 in the milk of the six transgenic goats ranged from 98 to 121 µg/ml at 15 days of lactation, and was maintained at 90–111 µg/ml during the following 2 months. Milk samples from transgenic goats showed an obvious inhibitory activity against E. coli, S. aureus and S. agalactiae in vitro. The minimal inhibitory concentrations of rHBD3 in milk against E. coli, S. aureus and S. agalactiae were 9.5–10.5, 21.8–23.0 and 17.3–18.5 µg/mL, respectively, which was similar to those of the HBD3 standard (P>0.05). The in vivo anti-bacterial activities of rHBD3 in milk were examined by intramammary infusion of viable bacterial inoculums. We observed that 9/10 and 8/10 glands of non-transgenic goats infused with S. aureus and E. coli became infected. The mean numbers of viable bacteria went up to 2.9×103 and 95.4×103 CFU/ml at 48 h after infusion, respectively; the mean somatic cell counts (SCC) in infected glands reached up to 260.4×105 and 622.2×105 cells/ml, which were significantly higher than the SCC in uninfected goat glands. In contrast, no bacteria was presented in glands of transgenic goats and PBS-infused controls, and the SSC did not significantly change throughout the period. Moreover, the compositions and protein profiles of milk from transgenic and non-transgenic goats were identical. The present study demonstrated that HBD3 were an effective anti-bacterial protein to enhance the mastitis resistance of dairy animals. PMID:23799010

  1. Anti-bacterial activity of recombinant human β-defensin-3 secreted in the milk of transgenic goats produced by somatic cell nuclear transfer.

    PubMed

    Liu, Jun; Luo, Yan; Ge, Hengtao; Han, Chengquan; Zhang, Hui; Wang, Yongsheng; Su, Jianmin; Quan, Fusheng; Gao, Mingqing; Zhang, Yong

    2013-01-01

    The present study was conducted to determine whether recombinant human β-defensin-3 (rHBD3) in the milk of transgenic goats has an anti-bacterial activity against Escherichia coli (E. coli), Staphylococcus aureus (S. aureus) and Streptococcus agalactiae (S. agalactiae) that could cause mastitis. A HBD3 mammary-specific expression vector was transfected by electroporation into goat fetal fibroblasts which were used to produce fourteen healthy transgenic goats by somatic cell nuclear transfer. The expression level of rHBD3 in the milk of the six transgenic goats ranged from 98 to 121 µg/ml at 15 days of lactation, and was maintained at 90-111 µg/ml during the following 2 months. Milk samples from transgenic goats showed an obvious inhibitory activity against E. coli, S. aureus and S. agalactiae in vitro. The minimal inhibitory concentrations of rHBD3 in milk against E. coli, S. aureus and S. agalactiae were 9.5-10.5, 21.8-23.0 and 17.3-18.5 µg/mL, respectively, which was similar to those of the HBD3 standard (P>0.05). The in vivo anti-bacterial activities of rHBD3 in milk were examined by intramammary infusion of viable bacterial inoculums. We observed that 9/10 and 8/10 glands of non-transgenic goats infused with S. aureus and E. coli became infected. The mean numbers of viable bacteria went up to 2.9×10(3) and 95.4×10(3) CFU/ml at 48 h after infusion, respectively; the mean somatic cell counts (SCC) in infected glands reached up to 260.4×10(5) and 622.2×10(5) cells/ml, which were significantly higher than the SCC in uninfected goat glands. In contrast, no bacteria was presented in glands of transgenic goats and PBS-infused controls, and the SSC did not significantly change throughout the period. Moreover, the compositions and protein profiles of milk from transgenic and non-transgenic goats were identical. The present study demonstrated that HBD3 were an effective anti-bacterial protein to enhance the mastitis resistance of dairy animals.

  2. Measuring micro-organism gas production

    NASA Technical Reports Server (NTRS)

    Wilkins, J. R.; Pearson, A. O.; Mills, S. M.

    1973-01-01

    Transducer, which senses pressure buildup, is easy to assemble and use, and rate of gas produced can be measured automatically and accurately. Method can be used in research, in clinical laboratories, and for environmental pollution studies because of its ability to detect and quantify rapidly the number of gas-producing microorganisms in water, beverages, and clinical samples.

  3. Micro-Organ Device

    NASA Technical Reports Server (NTRS)

    Gonda, Steve R. (Inventor); Chang, Robert C. (Inventor); Starly, Binil (Inventor); Culbertson, Christopher (Inventor); Holtorf, Heidi L. (Inventor); Sun, Wei (Inventor); Leslie, Julia (Inventor)

    2013-01-01

    A method for fabricating a micro-organ device comprises providing a microscale support having one or more microfluidic channels and one or more micro-chambers for housing a micro-organ and printing a micro-organ on the microscale support using a cell suspension in a syringe controlled by a computer-aided tissue engineering system, wherein the cell suspension comprises cells suspended in a solution containing a material that functions as a three-dimensional scaffold. The printing is performed with the computer-aided tissue engineering system according to a particular pattern. The micro-organ device comprises at least one micro-chamber each housing a micro-organ; and at least one microfluidic channel connected to the micro-chamber, wherein the micro-organ comprises cells arranged in a configuration that includes microscale spacing between portions of the cells to facilitate diffusion exchange between the cells and a medium supplied from the at least one microfluidic channel.

  4. Micro-organ device

    NASA Technical Reports Server (NTRS)

    Gonda, Steve R. (Inventor); von Gustedt-Gonda, legal representative, Iris (Inventor); Chang, Robert C. (Inventor); Starly, Binil (Inventor); Culbertson, Christopher (Inventor); Holtorf, Heidi L. (Inventor); Sun, Wei (Inventor); Leslie, Julia (Inventor)

    2013-01-01

    A method for fabricating a micro-organ device comprises providing a microscale support having one or more microfluidic channels and one or more micro-chambers for housing a micro-organ and printing a micro-organ on the microscale support using a cell suspension in a syringe controlled by a computer-aided tissue engineering system, wherein the cell suspension comprises cells suspended in a solution containing a material that functions as a three-dimensional scaffold. The printing is performed with the computer-aided tissue engineering system according to a particular pattern. The micro-organ device comprises at least one micro-chamber each housing a micro-organ; and at least one microfluidic channel connected to the micro-chamber, wherein the micro-organ comprises cells arranged in a configuration that includes microscale spacing between portions of the cells to facilitate diffusion exchange between the cells and a medium supplied from the at least one microfluidic channel.

  5. Recombinant zymomonas for pentose fermentation

    DOEpatents

    Picataggio, Stephen K.; Zhang, Min; Eddy, Christina K.; Deanda, Kristine A.; Finkelstein, Mark

    1996-01-01

    The invention relates to microorganisms which normally do not ferment a pentose sugar and which are genetically altered to ferment this pentose to produce ethanol. A representative example is Zymomonas mobilis which has been transformed with E. coli xylose isomerase, xylulokinase, transaldolase and transketolase genes. Expression of the added genes are under the control of Zymomonas mobilis promoters. This newly created microorganism is useful for fermenting pentoses and glucose, produced by hydrolysis of hemicellulose and cellulose, to produce ethanol.

  6. Recombinant Zymomonas for pentose fermentation

    DOEpatents

    Picataggio, S.K.; Zhang, M.; Eddy, C.K.; Deanda, K.A.; Finkelstein, M.

    1996-05-07

    The invention relates to microorganisms which normally do not ferment a pentose sugar and which are genetically altered to ferment this pentose to produce ethanol. A representative example is Zymomonas mobilis which has been transformed with E. coli xylose isomerase, xylulokinase, transaldolase and transketolase genes. Expression of the added genes are under the control of Zymomonas mobilis promoters. This newly created microorganism is useful for fermenting pentoses and glucose, produced by hydrolysis of hemicellulose and cellulose, to produce ethanol. 2 figs.

  7. Expanding the recombinant protein quality in Lactococcus lactis.

    PubMed

    Cano-Garrido, Olivia; Rueda, Fabian L; Sànchez-García, Laura; Ruiz-Ávila, Luis; Bosser, Ramon; Villaverde, Antonio; García-Fruitós, Elena

    2014-12-04

    Escherichia coli has been a main host for the production of recombinant proteins of biomedical interest, but conformational stress responses impose severe bottlenecks that impair the production of soluble, proteolytically stable versions of many protein species. In this context, emerging Generally Recognized As Safe (GRAS) bacterial hosts provide alternatives as cell factories for recombinant protein production, in which limitations associated to the use of Gram-negative microorganisms might result minimized. Among them, Lactic Acid Bacteria and specially Lactococcus lactis are Gram-positive GRAS organisms in which recombinant protein solubility is generically higher and downstream facilitated, when compared to E. coli. However, deep analyses of recombinant protein quality in this system are still required to completely evaluate its performance and potential for improvement. We have explored here the conformational quality (through specific fluorescence emission) and solubility of an aggregation-prone GFP variant (VP1GFP) produced in L. lactis. In this context, our results show that parameters such as production time, culture conditions and growth temperature have a dramatic impact not only on protein yield, but also on protein solubility and conformational quality, that are particularly favored under fermentative metabolism. Metabolic regime and cultivation temperature greatly influence solubility and conformational quality of an aggregation-prone protein in L. lactis. Specifically, the present study proves that anaerobic growth is the optimal condition for recombinant protein production purposes. Besides, growth temperature plays an important role regulating both protein solubility and conformational quality. Additionally, our results also prove the great versatility for the manipulation of this bacterial system regarding the improvement of functionality, yield and quality of recombinant proteins in this species. These findings not only confirm L. lactis as an

  8. Recombinant host cells and media for ethanol production

    SciTech Connect

    Wood, Brent E; Ingram, Lonnie O; Yomano, Lorraine P; York, Sean W

    2014-02-18

    Disclosed are recombinant host cells suitable for degrading an oligosaccharide that have been optimized for growth and production of high yields of ethanol, and methods of making and using these cells. The invention further provides minimal media comprising urea-like compounds for economical production of ethanol by recombinant microorganisms. Recombinant host cells in accordance with the invention are modified by gene mutation to eliminate genes responsible for the production of unwanted products other than ethanol, thereby increasing the yield of ethanol produced from the oligosaccharides, relative to unmutated parent strains. The new and improved strains of recombinant bacteria are capable of superior ethanol productivity and yield when grown under conditions suitable for fermentation in minimal growth media containing inexpensive reagents. Systems optimized for ethanol production combine a selected optimized minimal medium with a recombinant host cell optimized for use in the selected medium. Preferred systems are suitable for efficient ethanol production by simultaneous saccharification and fermentation (SSF) using lignocellulose as an oligosaccharide source. The invention also provides novel isolated polynucleotide sequences, polypeptide sequences, vectors and antibodies.

  9. Fossil Microorganisms in Archaean

    NASA Technical Reports Server (NTRS)

    Astafleva, Marina; Hoover, Richard; Rozanov, Alexei; Vrevskiy, A.

    2006-01-01

    Ancient Archean and Proterozoic rocks are the model objects for investigation of rocks comprising astromaterials. The first of Archean fossil microorganisms from Baltic shield have been reported at the last SPIE Conference in 2005. Since this confeence biomorphic structures have been revealed in Archean rocks of Karelia. It was determined that there are 3 types of such bion structures: 1. structures found in situ, in other words microorganisms even-aged with rock matrix, that is real Archean fossils biomorphic structures, that is to say forms inhabited early formed rocks, and 3. younger than Archean-Protherozoic minerali microorganisms, that is later contamination. We made attempt to differentiate these 3 types of findings and tried to understand of burial of microorganisms. The structures belongs (from our point of view) to the first type, or real Archean, forms were under examination. Practical investigation of ancient microorganisms from Green-Stone-Belt of Northern Karelia turns to be very perspective. It shows that even in such ancient time as Archean ancient diverse world existed. Moreover probably such relatively highly organized cyanobacteria and perhaps eukaryotic formes existed in Archean world.

  10. Fossil Microorganisms in Archaean

    NASA Technical Reports Server (NTRS)

    Astafleva, Marina; Hoover, Richard; Rozanov, Alexei; Vrevskiy, A.

    2006-01-01

    Ancient Archean and Proterozoic rocks are the model objects for investigation of rocks comprising astromaterials. The first of Archean fossil microorganisms from Baltic shield have been reported at the last SPIE Conference in 2005. Since this confeence biomorphic structures have been revealed in Archean rocks of Karelia. It was determined that there are 3 types of such bion structures: 1. structures found in situ, in other words microorganisms even-aged with rock matrix, that is real Archean fossils biomorphic structures, that is to say forms inhabited early formed rocks, and 3. younger than Archean-Protherozoic minerali microorganisms, that is later contamination. We made attempt to differentiate these 3 types of findings and tried to understand of burial of microorganisms. The structures belongs (from our point of view) to the first type, or real Archean, forms were under examination. Practical investigation of ancient microorganisms from Green-Stone-Belt of Northern Karelia turns to be very perspective. It shows that even in such ancient time as Archean ancient diverse world existed. Moreover probably such relatively highly organized cyanobacteria and perhaps eukaryotic formes existed in Archean world.

  11. Norovirus recombination.

    PubMed

    Bull, Rowena A; Tanaka, Mark M; White, Peter A

    2007-12-01

    RNA recombination is a significant driving force in viral evolution. Increased awareness of recombination within the genus Norovirus of the family Calicivirus has led to a rise in the identification of norovirus (NoV) recombinants and they are now reported at high frequency. Currently, there is no classification system for recombinant NoVs and a widely accepted recombinant genotyping system is still needed. Consequently, there is duplication in reporting of novel recombinants. This has led to difficulties in defining the number and types of recombinants in circulation. In this study, 120 NoV nucleotide sequences were compiled from the current GenBank database and published literature. NoV recombinants and their recombination breakpoints were identified using three methods: phylogenetic analysis, SimPlot analysis and the maximum chi2 method. A total of 20 NoV recombinant types were identified in circulation worldwide. The recombination point is the ORF1/2 overlap in all isolates except one, which demonstrated a double recombination event within the polymerase region.

  12. Alcohol fermentation of starch by a genetic recombinant yeast having glucoamylase activity.

    PubMed

    Nakamura, Y; Kobayashi, F; Ohnaga, M; Sawada, T

    1997-01-05

    Alcohol fermentation of starch was investigated using a direct starch fermenting yeast, Saccharomyces cerevisiae SR93, constructed by integrating a glucoamylase-producing gene (STA1) into the chromosome of Saccharomyces cerevisiae SH1089. The glucoamylase was constitutively produced by the recombinant yeast. The ethanol concentration produced by the recombinant yeast was 14.3 g/L which was about 1.5-fold higher than by the conventional mixed culture using an amylolytic microorganism and a fermenting microorganism. About 60% of the starch was converted into ethanol by the recombinant yeast, and the ethanol yield reached its maximum value of 0.48 at the initial starch concentration of 50 g/L. The fed-batch culture, which maintains the starch concentration in the range of 30 to 50 g/L, was used to produce a large amount of ethanol from starch. The amount of ethanol produced in the fed-batch culture increased about 20% compared to the batch culture. (c) 1997 John Wiley & Sons, Inc.

  13. Micro-Organ Devices

    NASA Technical Reports Server (NTRS)

    Gonda, Steven R.; Leslie, Julia; Chang, Robert C.; Starly, Binil; Sun, Wei; Culbertson, Christopher; Holtorf, Heidi

    2009-01-01

    Micro-organ devices (MODs) are being developed to satisfy an emerging need for small, lightweight, reproducible, biological-experimentati on apparatuses that are amenable to automated operation and that imp ose minimal demands for resources (principally, power and fluids). I n simplest terms, a MOD is a microfluidic device containing a variety of microstructures and assemblies of cells, all designed to mimic a complex in vivo microenvironment by replicating one or more in vivo micro-organ structures, the architectures and composition of the extr acellular matrices in the organs of interest, and the in vivo fluid flows. In addition to microscopic flow channels, a MOD contains one or more micro-organ wells containing cells residing in microscopic e xtracellular matrices and/or scaffolds, the shapes and compositions o f which enable replication of the corresponding in vivo cell assembl ies and flows.

  14. Elastohydrodynamics of flagellated microorganisms

    NASA Astrophysics Data System (ADS)

    Li, Gaojin; Ardekani, Arezoo

    2016-11-01

    The swimming motion of many microorganisms and cells are achieved by the waving deformation of their cilia and flagella. The typical structure of flagella and cilia contains nine doublets of parallel microtubules in a cylindrical arrangement surrounding one pair of microtubules in the center. The dynein molecular motors internally drive the sliding motion between the neighboring microtubules and cause the bending motion of the flagella and cilia and drive the microorganism swimming motion. In this work, we develop a numerical model for a microorganism swimming by an internally self-driven filament. Our numerical method captures the interaction between the elasticity of the flagellum and the surround fluid. The no-slip boundary conditions are satisfied by an iterative distributed Lagrangian multiplier method. We also investigate the effects of the non-Newtonian fluid rheology on the motion of an elastic flagellum near a wall.

  15. Bioplastics from microorganisms.

    PubMed

    Luengo, José M; García, Belén; Sandoval, Angel; Naharro, Germán; Olivera, Elías R

    2003-06-01

    The term 'biomaterials' includes chemically unrelated products that are synthesised by microorganisms (or part of them) under different environmental conditions. One important family of biomaterials is bioplastics. These are polyesters that are widely distributed in nature and accumulate intracellularly in microorganisms in the form of storage granules, with physico-chemical properties resembling petrochemical plastics. These polymers are usually built from hydroxy-acyl-CoA derivatives via different metabolic pathways. Depending on their microbial origin, bioplastics differ in their monomer composition, macromolecular structure and physical properties. Most of them are biodegradable and biocompatible, which makes them extremely interesting from the biotechnological point of view.

  16. Genomic homologous recombination in planta.

    PubMed Central

    Gal, S; Pisan, B; Hohn, T; Grimsley, N; Hohn, B

    1991-01-01

    A system for monitoring intrachromosomal homologous recombination in whole plants is described. A multimer of cauliflower mosaic virus (CaMV) sequences, arranged such that CaMV could only be produced by recombination, was integrated into Brassica napus nuclear DNA. This set-up allowed scoring of recombination events by the appearance of viral symptoms. The repeated homologous regions were derived from two different strains of CaMV so that different recombinant viruses (i.e. different recombination events) could be distinguished. In most of the transgenic plants, a single major virus species was detected. About half of the transgenic plants contained viruses of the same type, suggesting a hotspot for recombination. The remainder of the plants contained viruses with cross-over sites distributed throughout the rest of the homologous sequence. Sequence analysis of two recombinant molecules suggest that mismatch repair is linked to the recombination process. Images PMID:2026150

  17. Fermentations with new recombinant organisms

    SciTech Connect

    Bothast, R.J.; Nichols, N.N.; Dien, B.S.

    1999-10-01

    US fuel ethanol production in 1998 exceeded the record production of 1.4 billion gallons set in 1995. Most of this ethanol was produced from over 550 million bushels of corn. Expanding fuel ethanol production will require developing lower-cost feedstocks, and only lignocellulosic feedstocks are available in sufficient quantities to substitute for corn starch. Major technical hurdles to converting lignocellulose to ethanol include the lack of low-cost efficient enzymes for saccharification of biomass to fermentable sugars and the development of microorganisms for the fermentation of these mixed sugars. To date, the most successful research approaches to develop novel biocatalysts that will efficiently ferment mixed sugar syrups include isolation of novel yeasts that ferment xylose, genetic engineering of Escherichia coli and other gram negative bacteria for ethanol production, and genetic engineering of Saccharomyces cerevisiae and Zymomonas mobilis for pentose utilization. The authors have evaluated the fermentation of corn fiber hydrolyzates by the various strains developed. E. coli K011, E. coli SL40, E. coli FBR3, Zymomonas CP4 (pZB5), and Saccharomyces 1400 (pLNH32) fermented corn fiber hydrolyzates to ethanol in the range of 21--34 g/L with yields ranging from 0.41 to 0.50 g of ethanol per gram of sugar consumed. Progress with new recombinant microorganisms has been rapid and will continue with the eventual development of organisms suitable for commercial ethanol production. Each research approach holds considerable promise, with the possibility existing that different industrially hardened strains may find separate applications in the fermentation of specific feedstocks.

  18. Dissociative recombination in aeronomy

    NASA Technical Reports Server (NTRS)

    Fox, J. L.

    1989-01-01

    The importance of dissociative recombination in planetary aeronomy is summarized, and two examples are discussed. The first is the role of dissociative recombination of N2(+) in the escape of nitrogen from Mars. A previous model is updated to reflect new experimental data on the electronic states of N produced in this process. Second, the intensity of the atomic oxygen green line on the nightside of Venus is modeled. Use is made of theoretical rate coefficients for production of O (1S) in dissociative recombination from different vibrational levels of O2(+).

  19. Microorganism Utilization for Synthetic Milk Production

    NASA Technical Reports Server (NTRS)

    Birmele, Michele; Morford, Megan; Khodadad, Christina; Spencer, Lashelle; Richards, Jeffrey; Strayer, Richard; Caro, Janicce; Hummerick, Mary; Wheeler, Ray

    2014-01-01

    A desired architecture for long duration spaceflight, such as aboard the International Space Station (ISS) or for future missions to Mars, is to provide a supply of fresh food crops for the astronauts. However, some crops can create a high proportion of inedible plant waste. The main goal of this project was to produce the components of milk (sugar, lipid, protein) from inedible plant waste by utilizing microorganisms (fungi, yeast, bacteria). Of particular interest was utilizing the valuable polysaccharide, cellulose, found in plant waste, to naturally fuel- through microorganism cellular metabolism- the creation of sugar (glucose), lipid (milk fat), and protein (casein) to produce a synthetic edible food product. Environmental conditions such as pH, temperature, carbon source, aeration, and choice microorganisms.

  20. Biosurfactants, bioemulsifiers and exopolysaccharides from marine microorganisms.

    PubMed

    Satpute, Surekha K; Banat, Ibrahim M; Dhakephalkar, Prashant K; Banpurkar, Arun G; Chopade, Balu A

    2010-01-01

    Marine biosphere offers wealthy flora and fauna, which represents a vast natural resource of imperative functional commercial grade products. Among the various bioactive compounds, biosurfactant (BS)/bioemulsifiers (BE) are attracting major interest and attention due to their structural and functional diversity. The versatile properties of surface active molecules find numerous applications in various industries. Marine microorganisms such as Acinetobacter, Arthrobacter, Pseudomonas, Halomonas, Myroides, Corynebacteria, Bacillus, Alteromonas sp. have been studied for production of BS/BE and exopolysaccharides (EPS). Due to the enormity of marine biosphere, most of the marine microbial world remains unexplored. The discovery of potent BS/BE producing marine microorganism would enhance the use of environmental biodegradable surface active molecule and hopefully reduce total dependence or number of new application oriented towards the chemical synthetic surfactant industry. Our present review gives comprehensive information on BS/BE which has been reported to be produced by marine microorganisms and their possible potential future applications.

  1. Activated recombinant adenovirus proteinases

    DOEpatents

    Anderson, C.W.; Mangel, W.F.

    1999-08-10

    This application describes methods and expression constructs for producing activatable recombinant adenovirus proteinases. Purified activatable recombinant adenovirus proteinases and methods of purification are described. Activated adenovirus proteinases and methods for obtaining activated adenovirus proteinases are further included. Isolated peptide cofactors of adenovirus proteinase activity, methods of purifying and identifying peptide cofactors are also described. Antibodies immunoreactive with adenovirus proteinases, immunospecific antibodies, and methods for preparing them are also described. Other related methods and materials are also described. 29 figs.

  2. Activated recombinant adenovirus proteinases

    DOEpatents

    Anderson, Carl W.; Mangel, Walter F.

    1999-08-10

    This application describes methods and expression constructs for producing activatable recombinant adenovirus proteinases. Purified activatable recombinant adenovirus proteinases and methods of purification are described. Activated adenovirus proteinases and methods for obtaining activated adenovirus proteinases are further included. Isolated peptide cofactors of adenovirus proteinase activity, methods of purifying and identifying said peptide cofactors are also described. Antibodies immunoreactive with adenovirus proteinases, immunospecific antibodies, and methods for preparing them are also described. Other related methods and materials are also described.

  3. Microorganisms and Man.

    ERIC Educational Resources Information Center

    Noble, W. C.

    1983-01-01

    Provides information to update Institute of Biology's Studies in Biology No. 111, "Microorganisms and Man," by W. C. Noble and Jay Naidoo (Edward Arnold, 1979). Topics include: (1) food poisoning; (2) airborn infections in man; (3) infection in animals and plants; and (4) biodegradation and biosynthesis. (JN)

  4. Microorganisms and Man.

    ERIC Educational Resources Information Center

    Noble, W. C.

    1983-01-01

    Provides information to update Institute of Biology's Studies in Biology No. 111, "Microorganisms and Man," by W. C. Noble and Jay Naidoo (Edward Arnold, 1979). Topics include: (1) food poisoning; (2) airborn infections in man; (3) infection in animals and plants; and (4) biodegradation and biosynthesis. (JN)

  5. Recombinant synthesis of hyaluronan by Agrobacterium sp.

    PubMed

    Mao, Zichao; Chen, Rachel Ruizhen

    2007-01-01

    Hyaluronan (HA) is a sugar polymer of a repeating disaccharide, beta1-3 D-N-acetylglucosamine (GlcNAc) beta1-4 D-glucuronic acid (GlcA). It finds applications in numerous biomedical procedures such as ophthalmic surgery and osteoarthritis treatment. Until recently, the only commercial sources were extraction of rooster combs and from fermentation of pathogenic Streptococcus. In this work, we demonstrate that metabolic engineering strategies enable the recombinant synthesis of hyaluronan in a safe microorganism. Agrobacterium sp. ATCC 31749 is a commercial production strain for a food polymer, Curdlan. A broad host range expression vector was successfully developed to express the 3 kb HA synthase gene from Pasteurella multocida, along with a kfiD gene encoding UDP-glucose dehydrogenase from Escherichia coli K5 strain. Coexpression of these two heterologous enzymes enables Agrobacterium to produce HA. Hyaluronan was accumulated up to 0.3 g/L in shaker flask cultivation. The molecular weight of the polymer from various Agrobacterium strains is in the range of 0.7-2 MD. To our knowledge, this is the first successful recombinant hyaluronan synthesis in a Gram-negative bacterium that naturally produces a food product. The ease of genetic modifications provides future opportunities to tailor properties of polymers for specific applications.

  6. Evaluation of the ecotoxicity of pollutants with bioluminescent microorganisms.

    PubMed

    Fernández-Piñas, Francisca; Rodea-Palomares, Ismael; Leganés, Francisco; González-Pleiter, Miguel; Angeles Muñoz-Martín, M

    2014-01-01

    This chapter deals with the use of bioluminescent microorganisms in environmental monitoring, particularly in the assessment of the ecotoxicity of pollutants. Toxicity bioassays based on bioluminescent microorganisms are an interesting complement to classical toxicity assays, providing easiness of use, rapid response, mass production, and cost effectiveness. A description of the characteristics and main environmental applications in ecotoxicity testing of naturally bioluminescent microorganisms, covering bacteria and eukaryotes such as fungi and dinoglagellates, is reported in this chapter. The main features and applications of a wide variety of recombinant bioluminescent microorganisms, both prokaryotic and eukaryotic, are also summarized and critically considered. Quantitative structure-activity relationship models and hormesis are two important concepts in ecotoxicology; bioluminescent microorganisms have played a pivotal role in their development. As pollutants usually occur in complex mixtures in the environment, the use of both natural and recombinant bioluminescent microorganisms to assess mixture toxicity has been discussed. The main information has been summarized in tables, allowing quick consultation of the variety of luminescent organisms, bioluminescence gene systems, commercially available bioluminescent tests, environmental applications, and relevant references.

  7. Interventions for fresh produce

    USDA-ARS?s Scientific Manuscript database

    Environmental matrices such as soil, water, and dust harbor microorganisms. Many of the microorganisms found in the environment are essential for biogeochemical cycles and are essential for plant growth. The microbiome of the produce production environment might also contain foodborne pathogens and ...

  8. Application of recombinant human leukemia inhibitory factor (LIF) produced in rice (Oryza sativa L.) for maintenance of mouse embryonic stem cells.

    PubMed

    Youngblood, Bradford A; Alfano, Randall; Pettit, Steve C; Zhang, Deshui; Dallmann, H Garry; Huang, Ning; Macdonald, Clinton C

    2014-02-20

    Embryonic and induced pluripotent stem cells have the ability to differentiate into any somatic cell type, and thus have potential to treat a number of diseases that are currently incurable. Application of these cells for clinical or industrial uses would require an increase in production to yield adequate numbers of viable cells. However, the relatively high costs of cytokines and growth factors required for maintenance of stem cells in the undifferentiated state have the potential to limit translational research. Leukemia inhibitory factor (LIF), a member of the IL-6 cytokine family, is a key regulator in the maintenance of naïve states for both human and mouse stem cells. In this study, we describe a new recombinant human LIF (rhLIF) using a plant-based (rice) expression system. We found that rice-derived rhLIF possessed the same specific activity as commercial Escherichia coli-derived LIF and was capable of supporting mouse embryonic stem cell proliferation in the undifferentiated state as evidenced from pluripotency marker level analysis. Retention of the pluripotent state was found to be indistinguishable between rice-derived rhLIF and other recombinant LIF proteins currently on the market.

  9. Inactivation of Microorganisms

    NASA Astrophysics Data System (ADS)

    Alzamora, Stella Maris; Guerrero, Sandra N.; Schenk, Marcela; Raffellini, Silvia; López-Malo, Aurelio

    Minimal processing techniques for food preservation allow better retention of product flavor, texture, color, and nutrient content than comparable conventional treatments. A wide range of novel alternative physical factors have been intensely investigated in the last two decades. These physical factors can cause inactivation of microorganisms at ambient or sublethal temperatures (e.g., high hydrostatic pressure, pulsed electric fields, ultrasound, pulsed light, and ultraviolet light). These technologies have been reported to reduce microorganism population in foods while avoiding the deleterious effects of severe heating on quality. Among technologies, high-energy ultrasound (i.e., intensities higher than 1 W/cm2, frequencies between 18 and 100 kHz) has attracted considerable interest for food preservation applications (Mason et al., 1996; Povey and Mason, 1998).

  10. Environmentally relevant microorganisms.

    PubMed

    Watanabe, K; Baker, P W

    2000-01-01

    The development of molecular microbial ecology in the 1990s has allowed scientists to realize that microbial populations in the natural environment are much more diverse than microorganisms so far isolated in the laboratory. This finding has exerted a significant impact on environmental biotechnology, since knowledge in this field has been largely dependent on studies with pollutant-degrading bacteria isolated by conventional culture methods. Researchers have thus started to use molecular ecological methods to analyze microbial populations relevant to pollutant degradation in the environment (called environmentally relevant microorganisms, ERMs), although further effort is needed to gain practical benefits from these studies. This review highlights the utility and limitations of molecular ecological methods for understanding and advancing environmental biotechnology processes. The importance of the combined use of molecular ecological and physiological methods for identifying ERMs is stressed.

  11. Microorganisms and psoriasis.

    PubMed Central

    Rosenberg, E. W.; Noah, P. W.; Skinner, R. B.

    1994-01-01

    It has been suggested previously that psoriasis is best explained as a distinctive inflammatory response to a variety of microbial stimuli, all acting primarily through activation of the alternative complement pathway. For the past several years we have conducted a "Problem Psoriasis Clinic" based on that premise. Patients are questioned, examined, and subjected to microbiologic laboratory investigations in an attempt to identify possibly relevant microorganisms, and then are treated with antibiotics. This article lists the most commonly found microorganisms in psoriasis patients and describes the usual treatment for each. Results obtained with this approach compare favorably with those achieved with more usual anti-psoriasis treatments. We recommend that a microbiologic investigation and a trial of antimicrobial treatment should precede any plan to treat psoriasis patients with anything more than the simplest topical agents. PMID:8040907

  12. DNA sequences, recombinant DNA molecules and processes for producing the A and B subunits of cholera toxin and preparations containing so-obtained subunit or subunits

    SciTech Connect

    Harford, N.; De Wilde, M.

    1987-05-19

    A recombinant DNA molecule is described comprising at least a portion coding for subunits A and B of cholera toxin, or a fragment or derivative of the portion wherein the fragment or derivative codes for a polypeptide have an activity which can induce an immune response to subunit A; can induce an immune response to subunit A and cause epithelial cell penetration and the enzymatic effect leading to net loss of fluid into the gut lumen; can bind to the membrane receptor for the B subunit of cholera toxin; can induce an immune response to subunit B; can induce an immune response to subunit B and bind to the membrane receptor; or has a combination of the activities.

  13. High yield production of extracellular recombinant levansucrase by Bacillus megaterium.

    PubMed

    Korneli, Claudia; Biedendieck, Rebekka; David, Florian; Jahn, Dieter; Wittmann, Christoph

    2013-04-01

    In this study, a high yield production bioprocess with recombinant Bacillus megaterium for the production of the extracellular enzyme levansucrase (SacB) was developed. For basic optimization of culture parameters and nutrients, a recombinant B. megaterium reporter strain that produced green fluorescent protein under control of a vector-based xylose-inducible promoter was used. It enabled efficient microtiter plate-based screening via fluorescence analysis. A pH value of pH 6, 20 % of dissolved oxygen, 37 °C, and elevated levels of biotin (100 μg L(-1)) were found optimal with regard to high protein yield and reduced overflow metabolism. Among the different compounds tested, fructose and glycerol were identified as the preferred source of carbon. Subsequently, the settings were transferred to a B. megaterium strain recombinantly producing levansucrase SacB based on the plasmid-located xylose-inducible expression system. In shake flask culture under the optimized conditions, the novel strain already secreted the target enzyme in high amounts (14 U mL(-1) on fructose and 17.2 U mL(-1) on glycerol). This was further increased in high cell density fed-batch processes up to 55 U mL(-1), reflecting a levansucrase concentration of 0.52 g L(-1). This is 100-fold more than previous efforts for this enzyme in B. megaterium and more than 10-fold higher than reported values of other extracellular protein produced in this microorganism so far. The recombinant strain could also handle raw glycerol from biodiesel industry which provided the same amount and quality of the recombinant protein and suggests future implementation into existing biorefinery concepts.

  14. Mass Spectrometer for Airborne Micro-Organisms

    NASA Technical Reports Server (NTRS)

    Sinha, M. P.; Friedlander, S. K.

    1986-01-01

    Bacteria and other micro-organisms identified continously with aid of new technique for producing samples for mass spectrometer. Technique generates aerosol of organisms and feeds to spectrometer. Given species of organism produces characteristic set of peaks in mass spectrum and thereby identified. Technique useful for monitoring bacterial makeup in environmental studies and in places where cleanliness is essential, such as hospital operating rooms, breweries, and pharmaceutical plants.

  15. Mass Spectrometer for Airborne Micro-Organisms

    NASA Technical Reports Server (NTRS)

    Sinha, M. P.; Friedlander, S. K.

    1986-01-01

    Bacteria and other micro-organisms identified continously with aid of new technique for producing samples for mass spectrometer. Technique generates aerosol of organisms and feeds to spectrometer. Given species of organism produces characteristic set of peaks in mass spectrum and thereby identified. Technique useful for monitoring bacterial makeup in environmental studies and in places where cleanliness is essential, such as hospital operating rooms, breweries, and pharmaceutical plants.

  16. Consolidated bioprocessing method using thermophilic microorganisms

    DOEpatents

    Mielenz, Jonathan Richard

    2016-02-02

    The present invention is directed to a method of converting biomass to biofuel, and particularly to a consolidated bioprocessing method using a co-culture of thermophilic and extremely thermophilic microorganisms which collectively can ferment the hexose and pentose sugars produced by degradation of cellulose and hemicelluloses at high substrate conversion rates. A culture medium therefor is also provided as well as use of the methods to produce and recover cellulosic ethanol.

  17. Reexamining opportunities for therapeutic protein production in eukaryotic microorganisms.

    PubMed

    Matthews, Catherine B; Wright, Chapman; Kuo, Angel; Colant, Noelle; Westoby, Matthew; Love, J Christopher

    2017-11-01

    Antibodies are an important class of therapeutics and are predominantly produced in Chinese Hamster Ovary (CHO) cell lines. While this manufacturing platform is sufficiently productive to supply patient populations of currently approved therapies, it is unclear whether or not the current CHO platform can address two significant areas of need: affordable access to biologics for patients around the globe and production of unprecedented quantities needed for very large populations of patients. Novel approaches to recombinant protein production for therapeutic biologic products may be needed, and might be enabled by non-mammalian expression systems and recent advances in bioengineering. Eukaryotic microorganisms such as fungi, microalgae, and protozoa offer the potential to produce high-quality antibodies in large quantities. In this review, we lay out the current understanding of a wide range of species and evaluate based on theoretical considerations which are best poised to deliver a step change in cost of manufacturing and volumetric productivity within the next decade.Related article: http://onlinelibrary.wiley.com/doi/10.1002/bit.26383/full. © 2017 Wiley Periodicals, Inc.

  18. Butanol tolerance in microorganisms

    DOEpatents

    Bramucci, Michael G.; Nagarajan, Vasantha

    2016-03-01

    Provided herein are recombinant yeast host cells and methods for their use for production of fermentation products from a pyruvate utilizing pathway. Yeast host cells provided herein comprise reduced pyruvate decarboxylase activity and modified adenylate cyclase activity. In embodiments, yeast host cells provided herein comprise resistance to butanol and increased biomass production.

  19. Genetic Recombination

    ERIC Educational Resources Information Center

    Whitehouse, H. L. K.

    1973-01-01

    Discusses the mechanisms of genetic recombination with particular emphasis on the study of the fungus Sordaria brevicollis. The study of recombination is facilitated by the use of mutants of this fungus in which the color of the ascospores is affected. (JR)

  20. Genetic Recombination

    ERIC Educational Resources Information Center

    Whitehouse, H. L. K.

    1973-01-01

    Discusses the mechanisms of genetic recombination with particular emphasis on the study of the fungus Sordaria brevicollis. The study of recombination is facilitated by the use of mutants of this fungus in which the color of the ascospores is affected. (JR)

  1. Detecting the presence of microorganisms

    NASA Technical Reports Server (NTRS)

    Wilkins, Judd R. (Inventor); Stoner, Glenn E. (Inventor)

    1977-01-01

    The presence of microorganisms in a sample is determined by culturing microorganisms in a growth medium which is in contact with a measuring electrode and a reference electrode and detecting a change in potential between the electrodes caused by the presence of the microorganisms in the medium with a high impedance potentiometer.

  2. RNA polymerase gene, microorganism having said gene and the production of RNA polymerase by the use of said microorganism

    DOEpatents

    Kotani, Hirokazu; Hiraoka, Nobutsugu; Obayashi, Akira

    1991-01-01

    SP6 bacteriophage RNA polymerase is produced by cultivating a new microorganism (particularly new strains of Escherichia coli) harboring a plasmid that carries SP6 bacteriophage RNA polymerase gene and recovering SP6 bacteriophage RNA polymerase from the culture broth. SP6 bacteriophage RNA polymerase gene is provided as are new microorganisms harboring a plasmid that carries SP6 bacteriophage RNA polymerase gene.

  3. Antifungal and antibacterial activity of marine microorganisms.

    PubMed

    El Amraoui, B; El Amraoui, M; Cohen, N; Fassouane, A

    2014-03-01

    In order to explore marine microorganisms with pharmaceutical potential, marine bacteria, collected from different coastal areas of the Moroccan Atlantic Ocean, were previously isolated from seawater, sediment, marine invertebrates and seaweeds. The antimicrobial activities of these microorganisms were investigated against the pathogens involved in human pathologies. Whole cultures of 34 marine microorganisms were screened for antimicrobial activities using the method of agar diffusion against three Gram-positive bacteria, two Gram-negative bacteria, and against yeast. The results showed that among the 34 isolates studied, 28 (82%) strains have antimicrobial activity against at least one pathogen studied, 11 (32%) strains have antifungal activity and 24 (76%) strains are active against Gram-positive bacteria, while 21 (62%) strains are active against Gram-negative bacteria. Among isolates having antimicrobial activity, 14 were identified and were assigned to the genera Acinetobacter, Aeromonas, Alcaligenes, Bacillus, Chromobacterium, Enterococcus, Pantoea and Pseudomonas. Due to a competitive role for space and nutrient, the marine microorganisms can produce antibiotic substance; therefore, these marine microorganisms were expected to be potential resources of natural antibiotic products.

  4. Opportunities for renewable bioenergy using microorganisms.

    PubMed

    Rittmann, Bruce E

    2008-06-01

    Global warming can be slowed, and perhaps reversed, only when society replaces fossil fuels with renewable, carbon-neutral alternatives. The best option is bioenergy: the sun's energy is captured in biomass and converted to energy forms useful to modern society. To make a dent in global warming, bioenergy must be generated at a very high rate, since the world today uses approximately 10 TW of fossil-fuel energy. And, it must do so without inflicting serious damage on the environment or disrupting our food supply. While most bioenergy options fail on both counts, several microorganism-based options have the potential to produce large amounts of renewable energy without disruptions. In one approach, microbial communities convert the energy value of various biomass residuals to socially useful energy. Biomass residuals come from agricultural, animal, and a variety of industrial operations, as well as from human wastes. Microorganisms can convert almost all of the energy in these wastes to methane, hydrogen, and electricity. In a second approach, photosynthetic microorganisms convert sunlight into biodiesel. Certain algae (eukaryotes) or cyanobacteria (prokaryotes) have high lipid contents. Under proper conditions, these photosynthetic microorganisms can produce lipids for biodiesel with yields per unit area 100 times or more than possible with any plant system. In addition, the non-lipid biomass can be converted to methane, hydrogen, or electricity. Photosynthetic microorganisms do not require arable land, an advantage because our arable land must be used to produce food. Algae or cyanobacteria may be the best option to produce bioenergy at rates high enough to replace a substantial fraction of our society's use of fossil fuels.

  5. Hydrocortisone made in yeast: metabolic engineering turns a unicellular microorganism into a drug-synthesizing factory.

    PubMed

    Dumas, Bruno; Brocard-Masson, Corinne; Assemat-Lebrun, Karine; Achstetter, Tilman

    2006-03-01

    Inspired by the successful work of converting Saccharomyces cerevisiae into an microorganism capable of synthesizing hydrocortisone, a 27-carbon molecule, from ethanol, a 2-carbon molecule, this review provides an overview of the potential of yeast as a recombinant organism in the 21st century. Yeast has been used by man for more than 6,000 years, and is still paving the way to new discoveries. It was the first eukaryotic organism to be sequenced, in 1996, and the first to produce hydrocortisone in 2003. In addition, extensive genome-wide analyses have been performed with yeast. In this review, we discuss the pros and cons of using yeast to produce small therapeutic molecules. It is obvious that S. cerevisiae has a cutting edge advantage of being a well-known organism and time will tell if yeast "biohydrocortisone" is a unique example or the beginning of a long list of yeast bioproducts. Other organisms, such as plants and bacteria, are competing with yeast. Bacteria produce a wealth of marketed molecules and plants are capable of producing extremely complex molecules with an unbeatable yield. However, S. cerevisiae offers a unique mix of the simplicity of a recombinant organism combined with the complexity of a eukaryote.

  6. Two recombinant human interferon-beta 1a pharmaceutical preparations produce a similar transcriptional response determined using whole genome microarray analysis.

    PubMed

    Prync, A E Sterin; Yankilevich, P; Barrero, P R; Bello, R; Marangunich, L; Vidal, A; Criscuolo, M; Benasayag, L; Famulari, A L; Domínguez, R O; Kauffman, M A; Diez, R A

    2008-02-01

    Recombinant human interferon-beta (IFN-b) is a well-established treatment for multiple sclerosis (MS). The regulatory process for marketing authorization of biosimilars is currently under debate in certain countries. In the EU, EMEA has clearly defined the process including overarching and product-specific guidelines, which includes clinical testing. Biosimilarity needs to be based on comparability criteria, including at least molecular characterization, biological activity relevant for the therapeutic effect and relative bioavailability ("bioequivalence"). In the case of such complex diseases as MS, where the effect of treatment is not so directly measurable, in vitro tools can provide additional data to support comparability. Genomic microarrays assays might be useful to compare multisource biopharmaceuticals. The aim of the present study was to compare the pharmacodynamic genomic effects (in terms of transcriptional regulation) of two recombinant human IFN-I(2)1a preparations on lymphocytes of multiple sclerosis patients using a whole genome microarray assay. We performed an ex vivo whole genome expression profiling of the effect of two preparations of IFN-I(2)1a on non-adherent mononuclears from five relapsing-remitting MS patients analyzing microarrays (CodeLink Human Whole Genome). Patients blood was drawn, PBMCs isolated and cultured in three different conditions: culture medium (control), 1,000 U/ml of IFN-I(2)1a (BLA- (STOFERON, Bio Sidus) and 1,000 U/ml of IFN-I(2)1a (REBIF, Serono) RNA was purified from non-adherent cells (mostly lymphocytes), amplified and hybridized. Raw data were generated by CodeLink proprietary software. Data normalization, quality control and analysis of differential gene expression between treatments were done using linear model for microarray data. Functional annotation analysis of IFN-I(2)1a MS treatment transcription was done using DAVID. Out of the approximately 45,000 human sequences examined, no evidence of differential

  7. Heterologous expression of newly identified galectin-8 from sea urchin embryos produces recombinant protein with lactose binding specificity and anti-adhesive activity

    PubMed Central

    Karakostis, Kostantinos; Costa, Caterina; Zito, Francesca; Matranga, Valeria

    2015-01-01

    Galectin family members specifically bind beta-galactoside derivatives and are involved in different cellular events, including cell communication, signalling, apoptosis, and immune responses. Here, we report a tandem-repeat type galectin from the Paracentrotus lividus sea urchin embryo, referred to as Pl-GAL-8. The 933nt sequence encodes a protein of 34.73 kDa, containing the conserved HFNPRF and WGxExR motifs in the two highly similar carbohydrate-recognition domains (CRD). The three-dimensional protein structure model of the N-CRD confirms the high evolutionary conservation of carbohydrate binding sites. The temporal gene expression is regulated during development and transcripts localize at the tip of the archenteron at gastrula stage, in a subset of the secondary mesenchyme cells that differentiate into blastocoelar (immune) cells. Functional studies using a recombinant Pl-GAL-8 expressed in bacteria demonstrate its hemo-agglutinating activity on human red blood cells through the binding to lactose, as well as its ability in inhibiting the adhesion of human Hep-G2 cells to the substrate. The recent implications in autoimmune diseases and inflammatory disorders make Gal-8 an attractive candidate for therapeutic purposes. Our results offer a solid basis for addressing the use of the new Pl-GAL-8 in functional and applicative studies, respectively in the developmental and biomedical fields. PMID:26640155

  8. Lead Speciation in Microorganisms.

    PubMed

    Stewart, Theodora J

    2017-04-10

    The biogeochemical cycles of lead (Pb) have been largely affected by anthropogenic activities as a result of its high natural abundance and use over the centuries [1]. At sites more strongly impacted by urbanization [2] and mining [3], Pb is found at high nano to low micromolar concentrations in surface waters, and can be significantly higher in soil and sediment [4]. Microorganisms are found everywhere and their responses to Pb exposure can range from resistant to highly sensitive [5, 6]. These varying levels of toxicity can be attributed to the cellular handling of Pb, making it important to understand the role of intracellular Pb speciation for more accurate toxicity predictions.

  9. Extremely thermophilic microorganisms as metabolic engineering platforms for production of fuels and industrial chemicals

    PubMed Central

    Zeldes, Benjamin M.; Keller, Matthew W.; Loder, Andrew J.; Straub, Christopher T.; Adams, Michael W. W.; Kelly, Robert M.

    2015-01-01

    Enzymes from extremely thermophilic microorganisms have been of technological interest for some time because of their ability to catalyze reactions of industrial significance at elevated temperatures. Thermophilic enzymes are now routinely produced in recombinant mesophilic hosts for use as discrete biocatalysts. Genome and metagenome sequence data for extreme thermophiles provide useful information for putative biocatalysts for a wide range of biotransformations, albeit involving at most a few enzymatic steps. However, in the past several years, unprecedented progress has been made in establishing molecular genetics tools for extreme thermophiles to the point that the use of these microorganisms as metabolic engineering platforms has become possible. While in its early days, complex metabolic pathways have been altered or engineered into recombinant extreme thermophiles, such that the production of fuels and chemicals at elevated temperatures has become possible. Not only does this expand the thermal range for industrial biotechnology, it also potentially provides biodiverse options for specific biotransformations unique to these microorganisms. The list of extreme thermophiles growing optimally between 70 and 100°C with genetic toolkits currently available includes archaea and bacteria, aerobes and anaerobes, coming from genera such as Caldicellulosiruptor, Sulfolobus, Thermotoga, Thermococcus, and Pyrococcus. These organisms exhibit unusual and potentially useful native metabolic capabilities, including cellulose degradation, metal solubilization, and RuBisCO-free carbon fixation. Those looking to design a thermal bioprocess now have a host of potential candidates to choose from, each with its own advantages and challenges that will influence its appropriateness for specific applications. Here, the issues and opportunities for extremely thermophilic metabolic engineering platforms are considered with an eye toward potential technological advantages for high

  10. Extremely thermophilic microorganisms as metabolic engineering platforms for production of fuels and industrial chemicals.

    PubMed

    Zeldes, Benjamin M; Keller, Matthew W; Loder, Andrew J; Straub, Christopher T; Adams, Michael W W; Kelly, Robert M

    2015-01-01

    Enzymes from extremely thermophilic microorganisms have been of technological interest for some time because of their ability to catalyze reactions of industrial significance at elevated temperatures. Thermophilic enzymes are now routinely produced in recombinant mesophilic hosts for use as discrete biocatalysts. Genome and metagenome sequence data for extreme thermophiles provide useful information for putative biocatalysts for a wide range of biotransformations, albeit involving at most a few enzymatic steps. However, in the past several years, unprecedented progress has been made in establishing molecular genetics tools for extreme thermophiles to the point that the use of these microorganisms as metabolic engineering platforms has become possible. While in its early days, complex metabolic pathways have been altered or engineered into recombinant extreme thermophiles, such that the production of fuels and chemicals at elevated temperatures has become possible. Not only does this expand the thermal range for industrial biotechnology, it also potentially provides biodiverse options for specific biotransformations unique to these microorganisms. The list of extreme thermophiles growing optimally between 70 and 100°C with genetic toolkits currently available includes archaea and bacteria, aerobes and anaerobes, coming from genera such as Caldicellulosiruptor, Sulfolobus, Thermotoga, Thermococcus, and Pyrococcus. These organisms exhibit unusual and potentially useful native metabolic capabilities, including cellulose degradation, metal solubilization, and RuBisCO-free carbon fixation. Those looking to design a thermal bioprocess now have a host of potential candidates to choose from, each with its own advantages and challenges that will influence its appropriateness for specific applications. Here, the issues and opportunities for extremely thermophilic metabolic engineering platforms are considered with an eye toward potential technological advantages for high

  11. The recombination of genetic material

    SciTech Connect

    Low, K.B.

    1988-01-01

    Genetic recombination is the major mechanism by which new arrangements of genetic elements are produced in all living organisms, from the simplest bacterial viruses to humans. This volume presents an overview of the types of recombination found in prokaryotes and eukaryotes.

  12. Recombinant, rice-produced yeast phytase shows the ability to hydrolyze phytate derived from seed-based feed, and extreme stability during ensilage treatment.

    PubMed

    Hamada, Akira; Yamaguchi, Ken-Ichi; Harada, Michiko; Horiguchi, Ken-Ichi; Takahashi, Toshiyoshi; Honda, Hideo

    2006-06-01

    When fresh rice leaves producing yeast Schwanniomyces occidentalis phytase were grounded and mixed with the whole extract of seed-based feed for pigs, the release of orthophosphate increased significantly. More specifically, phytate, a major source of phosphorus in the seeds, was hydrolyzed by heterologous phytase. Moreover, when transgenic rice plants were ensiled for up to 12 weeks, no decrease in the phytase activity of the heterologous enzyme was observed. This result strongly suggests that transgenic rice plants producing yeast phytase can be stored as silage without any loss of enzyme activity until usage as a feed additive.

  13. New skin test for detection of bovine tuberculosis on the basis of antigen-displaying polyester inclusions produced by recombinant Escherichia coli.

    PubMed

    Chen, Shuxiong; Parlane, Natalie A; Lee, Jason; Wedlock, D Neil; Buddle, Bryce M; Rehm, Bernd H A

    2014-04-01

    The tuberculin skin test for diagnosing tuberculosis (TB) in cattle lacks specificity if animals are sensitized to environmental mycobacteria, as some antigens in purified protein derivative (PPD) prepared from Mycobacterium bovis are present in nonpathogenic mycobacteria. Three immunodominant TB antigens, ESAT6, CFP10, and Rv3615c, are present in members of the pathogenic Mycobacterium tuberculosis complex but absent from the majority of environmental mycobacteria. These TB antigens have the potential to enhance skin test specificity. To increase their immunogenicity, these antigens were displayed on polyester beads by translationally fusing them to a polyhydroxyalkanoate (PHA) synthase which mediated formation of antigen-displaying inclusions in recombinant Escherichia coli. The most common form of these inclusions is poly(3-hydroxybutyric acid) (PHB). The respective fusion proteins displayed on these PHB inclusions (beads) were identified using tryptic peptide fingerprinting analysis in combination with matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS). The surface exposure and accessibility of antigens were assessed by enzyme-linked immunosorbent assay (ELISA). Polyester beads displaying all three TB antigens showed greater reactivity with TB antigen-specific antibody than did beads displaying only one TB antigen. This was neither due to cross-reactivity of antibodies with the other two antigens nor due to differences in protein expression levels between beads displaying single or three TB antigens. The triple-antigen-displaying polyester beads were used for skin testing of cattle and detected all cattle experimentally infected with M. bovis with no false-positive reactions observed in those sensitized to environmental mycobacteria. The results suggested applicability of TB antigen-displaying polyester inclusions as diagnostic reagents for distinguishing TB-infected from noninfected animals.

  14. Introduction to dissociative recombination

    NASA Technical Reports Server (NTRS)

    Guberman, Steven L.; Mitchell, J. Brian A.

    1989-01-01

    Dissociative recombination (DR) of molecular ions with electrons has important consequences in many areas of physical science. Ab-initio calculations coupled with resonant scattering theory and multichannel quantum defect studies have produced detailed results illuminating the role of ion vibrational excitation, the quantum yields of the DR products, and the role of Rydberg states. The theoretical and experimental results are discussed.

  15. Microorganisms having enhanced resistance to acetate and methods of use

    DOEpatents

    Brown, Steven D; Yang, Shihui

    2014-10-21

    The present invention provides isolated or genetically modified strains of microorganisms that display enhanced resistance to acetate as a result of increased expression of a sodium proton antiporter. The present invention also provides methods for producing such microbial strains, as well as related promoter sequences and expression vectors. Further, the present invention provides methods of producing alcohol from biomass materials by using microorganisms with enhanced resistance to acetate.

  16. Marine Microorganisms: perspectives for getting involved in cellulosic ethanol

    PubMed Central

    2012-01-01

    The production of ethanol has been considered as an alternative to replace part of the petroleum derivate. Brazil and the US are the leading producers, but more environmentally friendly alternatives are needed. Lignocellulose has an enormous potential but technology has to be still improve in order to economically produce ethanol. The present paper reviews the potential and problems of this technology and proposes the study of a group of microorganisms with the largest genetic pool, marine microorganism. PMID:22931793

  17. Marine Microorganisms: perspectives for getting involved in cellulosic ethanol.

    PubMed

    Intriago, Pablo

    2012-08-29

    The production of ethanol has been considered as an alternative to replace part of the petroleum derivate. Brazil and the US are the leading producers, but more environmentally friendly alternatives are needed. Lignocellulose has an enormous potential but technology has to be still improve in order to economically produce ethanol. The present paper reviews the potential and problems of this technology and proposes the study of a group of microorganisms with the largest genetic pool, marine microorganism.

  18. Microorganism Utilization for Synthetic Milk Production

    NASA Technical Reports Server (NTRS)

    Morford, Megan A.; Khodadad, Christina Louise; Spencer, LaShelle E.; Richards, Jeffrey T.; Strayer, Richard F.; Caro, Janicce; Hummerick, Mary; Birmele, Michele N.; Wheeler, Raymond M.

    2014-01-01

    A desired architecture for long duration spaceflight, such as aboard the International Space Station (ISS) or for future missions to Mars, is to provide a supply of fresh food crops for the astronauts. However, some crops can create a high proportion of inedible plant waste. The main goal of this project was to produce the components of milk (sugar, lipid, protein) from inedible plant waste by utilizing microorganisms (fungi, yeast, bacteria). Of particular interest was utilizing the valuable polysaccharide, cellulose, found in plant waste, to naturally fuel- through microorganism cellular metabolism- the creation of sugar (glucose), lipid (milk fat), and protein (casein) to produce a synthetic edible food product. Environmental conditions such as pH, temperature, carbon source, aeration, and choice microorganisms were optimized in the laboratory and the desired end-products, sugars and lipids, were analyzed. Trichoderma reesei, a known cellulolytic fungus, was utilized to drive the production of glucose, with the intent that the produced glucose would serve as the carbon source for milk fat production and be a substitute for the milk sugar lactose. Lipid production would be carried out by Rhodosporidium toruloides, yeast known to accumulate those lipids that are typically found in milk fat. Results showed that glucose and total lipid content were below what was expected during this phase of experimentation. In addition, individual analysis of six fatty acids revealed that the percentage of each fatty acid was lower than naturally produced bovine milk. Overall, this research indicates that microorganisms could be utilized to breakdown inedible solid waste to produce useable products.

  19. Microorganism Utilization for Synthetic Milk Production

    NASA Technical Reports Server (NTRS)

    Morford, Megan A.; Khodadad, Christina L.; Mccoy, LaShelle E.; Richards, Jeffrey T.; Strayer, Richard F.; Caro, Janicce L.; Hummerick, Mary E.; Birmele, Michele N.; Wheeler, Raymond M.

    2014-01-01

    A desired architecture for long duration spaceflight, such as aboard the International Space Station (ISS) or for future missions to Mars, is to provide a supply of fresh food crops for the astronauts. However, some crops can create a high proportion of inedible plant waste. The main goal of this project was to produce the components of milk (sugar, lipid, protein) from inedible plant waste by utilizing microorganisms (fungi, yeast, bacteria). Of particular interest was utilizing the valuable polysaccharide, cellulose, found in plant waste, to naturally fuel- through microorganism cellular metabolism- the creation of sugar (glucose), lipid (milk fat), and protein (casein) to produce a synthetic edible food product. Environmental conditions such as pH, temperature, carbon source, aeration, and choice microorganisms were optimized in the laboratory and the desired end-products, sugars and lipids, were analyzed. Trichoderma reesei, a known cellulolytic fungus, was utilized to drive the production of glucose, with the intent that the produced glucose would serve as the carbon source for milk fat production and be a substitute for the milk sugar lactose. Lipid production would be carried out by Rhodosporidium toruloides, yeast known to accumulate those lipids that are typically found in milk fat. Results showed that glucose and total lipid content were below what was expected during this phase of experimentation. In addition, individual analysis of six fatty acids revealed that the percentage of each fatty acid was lower than naturally produced bovine milk. Overall, this research indicates that microorganisms could be utilized to breakdown inedible solid waste to produce useable products.

  20. Biosynthesis of trans-4-hydroxyproline by recombinant strains of Corynebacterium glutamicum and Escherichia coli

    PubMed Central

    2014-01-01

    Background Trans-4-hydroxy-L-proline (trans-Hyp), one of the hydroxyproline (Hyp) isomers, is a useful chiral building block in the production of many pharmaceuticals. Although there are some natural biosynthetic pathways of trans-Hyp existing in microorganisms, the yield is still too low to be scaled up for industrial applications. Until now the production of trans-Hyp is mainly from the acid hydrolysis of collagen. Due to the increasing environmental concerns on those severe chemical processes and complicated downstream separation, it is essential to explore some environment-friendly processes such as constructing new recombinant strains to develop efficient process for trans-Hyp production. Result In this study, the genes of trans-proline 4-hydroxylase (trans-P4H) from diverse resources were cloned and expressed in Corynebacterium glutamicum and Escherichia coli, respectively. The trans-Hyp production by these recombinant strains was investigated. The results showed that all the genes from different resources had been expressed actively. Both the recombinant C. glutamicum and E. coli strains could produce trans-Hyp in the absence of proline and 2-oxoglutarate. Conclusions The whole cell microbial systems for trans-Hyp production have been successfully constructed by introducing trans-P4H into C. glutamicum and E. coli. Although the highest yield was obtained in recombinant E. coli, using recombinant C. glutamicum strains to produce trans-Hyp was a new attempt. PMID:24885047

  1. Recombinant Zymomonas for pentose fermentation

    DOEpatents

    Picataggio, S.K.; Min Zhang; Eddy, C.K.; Deanda, K.A.

    1998-03-10

    The invention relates to microorganisms which normally do not ferment pentose sugar and which are genetically altered to ferment pentose sugar to produce ethanol, and fermentation processes utilizing the same. Examples include Zymomonas mobilis which has been transformed with combinations of E. coli genes for xylose isomerase, xylulokinase, transaldolase, transketolase, L-arabinose isomerase, L-ribulokinase, and L-ribulose-5-phosphate 4-epimerase. Expression of the added genes are under the control of Zymomonas mobilis promoters. These newly created microorganisms are useful for fermenting pentoses and glucose, produced by hydrolysis of hemicellulose and cellulose, to produce ethanol. 7 figs.

  2. Pentose fermentation by recombinant Zymomonas

    DOEpatents

    Picataggio, S.K.; Zhang, M.; Eddy, C.K.; Deanda, K.A.; Finkelstein, M.; Mohagheghi, A.; Newman, M.M.; McMillan, J.D.

    1998-01-27

    The invention relates to microorganisms which normally do not ferment pentose sugar and which are genetically altered to ferment pentose sugar to produce ethanol, and fermentation processes utilizing the same. Examples include Zymomonas mobilis which has been transformed with combinations of E. coli genes for xylose isomerase, xylulokinase, transaldolase, transketolase, L-arabinose isomerase, L-ribulokinase, and L-ribulose 5-phosphate 4-epimerase. Expression of the added genes are under the control of Zymomonas mobilis promoters. These newly created microorganisms are useful for fermenting pentoses and glucose, produced by hydrolysis of hemicellulose and cellulose, to produce ethanol. 7 figs.

  3. Recombinant Zymomonas for pentose fermentation

    DOEpatents

    Picataggio, Stephen K.; Zhang, Min; Eddy, Christina K.; Deanda, Kristine A.

    1998-01-01

    The invention relates to microorganisms which normally do not ferment pentose sugar and which are genetically altered to ferment pentose sugar to produce ethanol, and fermentation processes utilizing the same. Examples include Zymomonas mobilis which has been transformed with combinations of E. coli genes for xylose isomerase, xylulokinase, transaldolase, transketolase, L-arabinose isomerase, L-ribulokinase, and L-ribulose-5-phosphate 4-epimerase. Expression of the added genes are under the control of Zymomonas mobilis promoters. These newly created microorganisms are useful for fermenting pentoses and glucose, produced by hydrolysis of hemicellulose and cellulose, to produce ethanol.

  4. Pentose fermentation by recombinant zymomonas

    DOEpatents

    Picataggio, Stephen K.; Zhang, Min; Eddy, Christina K.; Deanda, Kristine A.; Finkelstein, Mark; Mohagheghi, Ali; Newman, Mildred M.; McMillan, James D.

    1998-01-01

    The invention relates to microorganisms which normally do not ferment pentose sugar and which are genetically altered to ferment pentose sugar to produce ethanol, and fermentation processes utilizing the same. Examples include Zymomonas mobilis which has been transformed with combinations of E. coli genes for xylose isomerase, xylulokinase, transaldolase, transketolase, L-arabinose isomerase, L-ribulokinase, and L-ribulose 5-phosphate 4-epimerase. Expression of the added genes are under the control of Zymomonas mobilis promoters. These newly created microorganisms are useful for fermenting pentoses and glucose, produced by hydrolysis of hemicellulose and cellulose, to produce ethanol.

  5. The fibrous form of intracellular inclusion bodies in recombinant variant fibrinogen-producing cells is specific to the hepatic fibrinogen storage disease-inducible variant fibrinogen.

    PubMed

    Arai, Shinpei; Ogiwara, Naoko; Mukai, Saki; Takezawa, Yuka; Sugano, Mitsutoshi; Honda, Takayuki; Okumura, Nobuo

    2017-06-01

    Fibrinogen storage disease (FSD) is a rare disorder that is characterized by the accumulation of fibrinogen in hepatocytes and induces liver injury. Six mutations in the γC domain (γG284R, γT314P, γD316N, the deletion of γG346-Q350, γG366S, and γR375W) have been identified for FSD. Our group previously established γ375W fibrinogen-producing Chinese hamster ovary (CHO) cells and observed aberrant large granular and fibrous forms of intracellular inclusion bodies. The aim of this study was to investigate whether fibrous intracellular inclusion bodies are specific to FSD-inducible variant fibrinogen. Thirteen expression vectors encoding the variant γ-chain were stably or transiently transfected into CHO cells expressing normal fibrinogen Aα- and Bβ-chains or HuH-7 cells, which were then immunofluorescently stained. Six CHO and HuH-7 cell lines that transiently produced FSD-inducible variant fibrinogen presented the fibrous (3.2-22.7 and 2.1-24.5%, respectively) and large granular (5.4-25.5 and 7.7-23.9%) forms of intracellular inclusion bodies. Seven CHO and HuH-7 cell lines that transiently produced FSD-non-inducible variant fibrinogen only exhibit the large granular form. These results demonstrate that transiently transfected variant fibrinogen-producing CHO cells and inclusion bodies of the fibrous form may be useful in non-invasive screening for FSD risk factors for FSD before its onset.

  6. Toolbox for Antibiotics Discovery from Microorganisms.

    PubMed

    Fisch, Katja M; Schäberle, Till F

    2016-09-01

    Microorganisms produce a vast array of biologically active metabolites. Such compounds are applied by humans to positively influence their health and, therefore, natural products serve as drug leads for pharmaceutical and medicinal chemistry. In this minireview, tools for the discovery and the production of potential drug leads are explained. A snapshot is provided, starting from the isolation of new producer strains, across genomic mining of (meta)genomes to identify biosynthetic gene clusters corresponding to natural products, toward heterologous expression to produce potential drug leads. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  7. Making recombinant extracellular matrix proteins.

    PubMed

    Ruggiero, Florence; Koch, Manuel

    2008-05-01

    A variety of approaches to understand extracellular matrix protein structure and function require production of recombinant proteins. Moreover, the expression of heterologous extracellular matrix proteins, in particular collagens, using the recombinant technology is of major interest to the biomedical industry. Although extracellular matrix proteins are large, modular and often multimeric, most of them have been successfully produced in various expression systems. This review provides important factors, including the design of the construct, the cloning strategies, the expression vectors, the transfection method and the host cell systems, to consider in choosing a reliable and cost-effective way to make recombinant extracellular matrix proteins. Advantages and drawbacks of each system have been appraised. Protocols that may ease efficient recombinant production of extracellular matrix are described. Emphasis is placed on the recombinant collagen production. Members of the collagen superfamily exhibit specific structural features and generally require complex post-translational modifications to retain full biological activity that make more arduous their recombinant production.

  8. Production of cellulosic ethanol and enzyme from waste fiber sludge using SSF, recycling of hydrolytic enzymes and yeast, and recombinant cellulase-producing Aspergillus niger.

    PubMed

    Cavka, Adnan; Alriksson, Björn; Rose, Shaunita H; van Zyl, Willem H; Jönsson, Leif J

    2014-08-01

    Bioethanol and enzymes were produced from fiber sludges through sequential microbial cultivations. After a first simultaneous saccharification and fermentation (SSF) with yeast, the bioethanol concentrations of sulfate and sulfite fiber sludges were 45.6 and 64.7 g/L, respectively. The second SSF, which included fresh fiber sludges and recycled yeast and enzymes from the first SSF, resulted in ethanol concentrations of 38.3 g/L for sulfate fiber sludge and 24.4 g/L for sulfite fiber sludge. Aspergillus niger carrying the endoglucanase-encoding Cel7B gene of Trichoderma reesei was grown in the spent fiber sludge hydrolysates. The cellulase activities obtained with spent hydrolysates of sulfate and sulfite fiber sludges were 2,700 and 2,900 nkat/mL, respectively. The high cellulase activities produced by using stillage and the significant ethanol concentrations produced in the second SSF suggest that onsite enzyme production and recycling of enzyme are realistic concepts that warrant further attention.

  9. [Sherry wine microorganisms].

    PubMed

    García Maiquez, E

    1995-03-01

    Sherry wine presents, during all its wine-making and aging process, a great diversity of yeast and bacteria, as well as in the wine itself; its particular wine-making system, with traditional and legal additions to correct the acidity and to get a final alcoholic content of 15%, originates a selection of accompanying microorganisms. Species of the genera Kloeckera, Candida, Saccharomyces, Pichia, Hansenula and Saccharomycodes, have been isolated during the fermentation process in different proportions. This fact confirms that, besides S. cerevisiae, strains of S. chevalieri and S. fermentati have an important role in the fermentative process, and that the film-forming Saccharomyces have great activity in the fermentation. The biological aging of the Sherry wine, carried out by S. cheresiensis, S. beticus, S. feduchii and S. rouxii, has been studied in "finos" and "manzanillas". Different species and percentages in both wines have been described.

  10. Proteolysis in hyperthermophilic microorganisms

    DOE PAGES

    Ward, Donald E.; Shockley, Keith R.; Chang, Lara S.; ...

    2002-01-01

    Proteases are found in every cell, where they recognize and break down unneeded or abnormal polypeptides or peptide-based nutrients within or outside the cell. Genome sequence data can be used to compare proteolytic enzyme inventories of different organisms as they relate to physiological needs for protein modification and hydrolysis. In this review, we exploit genome sequence data to compare hyperthermophilic microorganisms from the euryarchaeotal genus Pyrococcus , the crenarchaeote Sulfolobus solfataricus , and the bacterium Thermotoga maritima . An overview of the proteases in these organisms is given based on those proteases that have been characterized and on putativemore » proteases that have been identified from genomic sequences, but have yet to be characterized. The analysis revealed both similarities and differences in the mechanisms utilized for proteolysis by each of these hyperthermophiles and indicated how these mechanisms relate to proteolysis in less thermophilic cells and organisms.« less

  11. Gravitaxis in unicellular microorganisms

    NASA Astrophysics Data System (ADS)

    Häder, D.-P.

    1999-01-01

    Orientation of organisms with respect to the gravitational field of the Earth has been studied for more than 100 years in a number of unicellular microorganisms including flagellates and ciliates. Several hypotheses have been developed how the weak stimulus is perceived. Intracellular statoliths have been found to be involved in gravitaxis of Loxodes, while no specialized organelles have been detected in other ciliates, e.g. Paramecium. Also in the slime mold Physarum no specialized gravireceptors have been identified yet. In the flagellate Euglena gracilis the whole cell body, which is denser than the surrounding medium, seems to act as a statolith pressing onto the lower membrane where it activates mechanosensitive ion channels. Similar results were obtained for the ciliate Paramecium. In contrast to the flagellate Euglena, several ciliates have been found to show gravikinesis, which is defined as a dependence of the swimming velocity on the direction of movement in the gravity field.

  12. Thermophilic microorganisms in biomining.

    PubMed

    Donati, Edgardo Rubén; Castro, Camila; Urbieta, María Sofía

    2016-11-01

    Biomining is an applied biotechnology for mineral processing and metal extraction from ores and concentrates. This alternative technology for recovering metals involves the hydrometallurgical processes known as bioleaching and biooxidation where the metal is directly solubilized or released from the matrix for further solubilization, respectively. Several commercial applications of biomining can be found around the world to recover mainly copper and gold but also other metals; most of them are operating at temperatures below 40-50 °C using mesophilic and moderate thermophilic microorganisms. Although biomining offers an economically viable and cleaner option, its share of the world´s production of metals has not grown as much as it was expected, mainly considering that due to environmental restrictions in many countries smelting and roasting technologies are being eliminated. The slow rate of biomining processes is for sure the main reason of their poor implementation. In this scenario the use of thermophiles could be advantageous because higher operational temperature would increase the rate of the process and in addition it would eliminate the energy input for cooling the system (bioleaching reactions are exothermic causing a serious temperature increase in bioreactors and inside heaps that adversely affects most of the mesophilic microorganisms) and it would decrease the passivation of mineral surfaces. In the last few years many thermophilic bacteria and archaea have been isolated, characterized, and even used for extracting metals. This paper reviews the current status of biomining using thermophiles, describes the main characteristics of thermophilic biominers and discusses the future for this biotechnology.

  13. A recombinant multi-antigen vaccine formulation containing Babesia bovis merozoite surface antigens MSA-2a1, MSA-2b and MSA-2c elicits invasion-inhibitory antibodies and IFN-γ producing cells.

    PubMed

    Gimenez, Alba Marina; Françoso, Katia S; Ersching, Jonatan; Icimoto, Marcelo Y; Oliveira, Vitor; Rodriguez, Anabel E; Schnittger, Leonhard; Florin-Christensen, Monica; Rodrigues, Mauricio M; Soares, Irene S

    2016-11-14

    Babesia bovis is a tick-transmitted protozoan hemoparasite and the causative agent of bovine babesiosis, a potential risk to more than 500 million cattle worldwide. The vaccines currently available are based on attenuated parasites, which are difficult to produce, and are only recommended for use in bovines under one year of age. When used in older animals, these vaccines may cause life-threatening clinical symptoms and eventually death. The development of a multi-subunit recombinant vaccine against B. bovis would be attractive from an economic standpoint and, most importantly, could be recommended for animals of any age. In the present study, recombinant ectodomains of MSA-2a1, MSA-2b and MSA-2c antigens were expressed in Pichia pastoris yeast as secreted soluble peptides. The antigens were purified to homogeneity, and biochemically and immunologically characterized. A vaccine formulation was obtained by emulsifying a mixture of the three peptides with the adjuvant Montanide ISA 720, which elicited high IgG antibody titers against each of the above antigens. IgG antibodies generated against each MSA-antigen recognized merozoites and significantly inhibited the invasion of bovine erythrocytes. Cellular immune responses were also detected, which were characterized by splenic and lymph node CD4(+) T cells producing IFN-γ and TNF-α upon stimulation with the antigens MSA-2a1 or MSA-2c. These data strongly suggest the high protective potential of the presented formulation, and we propose that it could be tested in vaccination trials of bovines challenged with B. bovis.

  14. Radiation sensitivity of hyperthermal composting microorganisms

    NASA Astrophysics Data System (ADS)

    Choi, Jong-Il; Yoon, Min-Chul; Kim, Jae-Hun; Yamashita, Masamichi; Kim, Geun Joong; Lee, Ju-Woon

    In the space station and vehicles designed for long human mission, high-temperature compost is a promising technology for decomposing organic waste and producing the fertilizers. In space, the microorganisms could have the changed biological activities or even be mutated by ionizing irradiation. Therefore, in this study, the effect of gamma irradiation on the sensitivity of bacteria in hyperthermal composting was investigated. The sequence analysis of the amplified 16s rDNA genes and amoA gene were used for the identification of composting microorganisms. Viability of microorganisms in compost soil after gamma irradiation was directly visualized with LIVE/DEAD Baclight viability kit. The dominant bacterial genera are Weissella cibaria and Leuconostoc sp. and fungus genera are Metschnikowia bicuspidate and Pichia guilliermondii, respectively. By the gamma irradiation up to the dose of 1 kGy, the microbial population was not changed. Also, the enzyme activities of amylase and cellulose were sustained by the gamma irradiation. These results show that these hyperthermia microorganisms might have the high resistance to gamma radiation and could be used for agriculture in the Space Station.

  15. Microorganism lipid droplets and biofuel development.

    PubMed

    Liu, Yingmei; Zhang, Congyan; Shen, Xipeng; Zhang, Xuelin; Cichello, Simon; Guan, Hongbin; Liu, Pingsheng

    2013-12-01

    Lipid droplet (LD) is a cellular organelle that stores neutral lipids as a source of energy and carbon. However, recent research has emerged that the organelle is involved in lipid synthesis, transportation, and metabolism, as well as mediating cellular protein storage and degradation. With the exception of multi-cellular organisms, some unicellular microorganisms have been observed to contain LDs. The organelle has been isolated and characterized from numerous organisms. Triacylglycerol (TAG) accumulation in LDs can be in excess of 50% of the dry weight in some microorganisms, and a maximum of 87% in some instances. These microorganisms include eukaryotes such as yeast and green algae as well as prokaryotes such as bacteria. Some organisms obtain carbon from CO2 via photosynthesis, while the majority utilizes carbon from various types of biomass. Therefore, high TAG content generated by utilizing waste or cheap biomass, coupled with an efficient conversion rate, present these organisms as bio-tech 'factories' to produce biodiesel. This review summarizes LD research in these organisms and provides useful information for further LD biological research and microorganism biodiesel development.

  16. Continuing fascination of exploration in natural substances from microorganisms.

    PubMed

    Takahashi, Yoko

    2017-01-01

    In the search for novel organic compounds, I think it is of paramount importance not to overlook the pursuit of microorganism diversity and the abilities those microorganisms hold as a resource. In commemoration of Professor Satoshi Ōmura's Nobel Prize in Physiology or Medicine, I will briefly describe the microorganism that produces avermectin and then discuss how innovating isolation methods and pioneering isolation sources have opened the door to numerous new microorganism resources. Furthermore, as exploratory research of substances views the world from many different angles-from biological activity to a compound's physiochemical properties-it is possible to discover a novel compound from a well-known microorganism. Based on this, I will discuss the future prospects of exploratory research.

  17. Identification, characterization, and recombinant expression of epidermicin NI01, a novel unmodified bacteriocin produced by Staphylococcus epidermidis that displays potent activity against Staphylococci.

    PubMed

    Sandiford, Stephanie; Upton, Mathew

    2012-03-01

    We describe the discovery, purification, characterization, and expression of an antimicrobial peptide, epidermicin NI01, which is an unmodified bacteriocin produced by Staphylococcus epidermidis strain 224. It is a highly cationic, hydrophobic, plasmid-encoded peptide that exhibits potent antimicrobial activity toward a wide range of pathogenic Gram-positive bacteria including methicillin-resistant Staphylococcus aureus (MRSA), enterococci, and biofilm-forming S. epidermidis strains. Purification of the peptide was achieved using a combination of hydrophobic interaction, cation exchange, and high-performance liquid chromatography (HPLC). Matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) analysis yielded a molecular mass of 6,074 Da, and partial sequence data of the peptide were elucidated using a combination of tandem mass spectrometry (MS/MS) and de novo sequencing. The draft genome sequence of the producing strain was obtained using 454 pyrosequencing technology, thus enabling the identification of the structural gene using the de novo peptide sequence data previously obtained. Epidermicin NI01 contains 51 residues with four tryptophan and nine lysine residues, and the sequence showed approximately 50% identity to peptides lacticin Z, lacticin Q, and aureocin A53, all of which belong to a new family of unmodified type II-like bacteriocins. The peptide is active in the nanomolar range against S. epidermidis, MRSA isolates, and vancomycin-resistant enterococci. Other unique features displayed by epidermicin include a high degree of protease stability and the ability to retain antimicrobial activity over a pH range of 2 to 10, and exposure to the peptide does not result in development of resistance in susceptible isolates. In this study we also show the structural gene alone can be cloned into Escherichia coli strain BL21(DE3), and expression yields active peptide.

  18. Recombinant allergens

    PubMed Central

    Jutel, Marek; Solarewicz-Madejek, Katarzyna; Smolinska, Sylwia

    2012-01-01

    Allergen specific immunotherapy (SIT) is the only known causative treatment of allergic diseases. Recombinant allergen-based vaccination strategies arose from a strong need to both to improve safety and enhance efficacy of SIT. In addition, new vaccines can be effective in allergies including food allergy or atopic dermatitis, which poorly respond to the current treatment with allergen extracts. A number of successful clinical studies with both wild-type and hypoallergenic derivatives of recombinant allergens vaccines have been reported for the last decade. They showed high efficacy and safety profile as well as very strong modulation of T and B cell responses to specific allergens. PMID:23095874

  19. Microorganisms in honey.

    PubMed

    Snowdon, J A; Cliver, D O

    1996-08-01

    Knowledge of the moisture and temperature conditions influencing growth of microorganisms in honey has long been used to control the spoilage of honey. However, the need for additional microbiological data on honey will increase as new technologies for, and uses of honey develop. Microorganisms in honey may influence quality or safety. Due to the natural properties of honey and control measures in the honey industry, honey is a product with minimal types and levels of microbes. Microbes of concern in post-harvest handling are those that are commonly found in honey (i.e., yeasts and spore-forming bacteria), those that indicate the sanitary or commercial quality of honey (i.e., coliforms and yeasts), and those that under certain conditions could cause human illness. Primary sources of microbial contamination are likely to include pollen, the digestive tracts of honey bees, dust, air, earth and nectar, sources which are very difficult to control. The same secondary (after-harvest) sources that influence any food product are also sources of contamination for honey. These include air, food handlers, cross-contamination, equipment and buildings. Secondary sources of contamination are controlled by good manufacturing practices. The microbes of concern in honey are primarily yeasts and spore-forming bacteria. Total plate counts from honey samples can vary from zero to tens of thousands per gram for no apparent reason. Most samples of honey contain detectable levels of yeasts. Although yeast counts in many honey samples are below 100 colony forming units per gram (cfu/g), yeasts can grow in honey to very high numbers. Standard industry practices control yeast growth. Bacterial spores, particularly those in the Bacillus genus, are regularly found in honey. The spores of C. botulinum are found in a fraction of the honey samples tested-normally at low levels. No vegetative forms of disease-causing bacterial species have been found in honey. Bacteria do not replicate in honey

  20. Population dynamics of transgenic microorganisms in the different microecosystem conditions

    NASA Astrophysics Data System (ADS)

    Popova, L. Yu.; Lobova, T. I.; Krylova, T. Yu.; Kargatova, T. V.; Maksimova, E. E.; Boyandin, A. N.; Pechurkin, N. S.

    The role of key environmental factors in adaptation of spore-forming and non-spore-forming transgenic microorganisms (TM) have been studied in model ecosystems. Model TM Escherichia coli Z905 (bearing plasmid genes of bacterial luminescence Ap rLux +) has been found to have a higher adaptation potential than TM Bacillus subtilis 2335/105 (bearing genes of human α 2-interferon Km rInf +), planned for employment as a living vaccine under varying environmental conditions. Effects of abiotic factors on migration of natural and recombinant plasmids between microorganisms under model ecosystem conditions has been estimated. The transgenic microorganisms with low copy number survived better under introduction conditions in the microcosms studied. This trend has been shown to be independent of the microcosm type and its complexity.

  1. A BioDesign Approach to Obtain High Yields of Biosimilars by Anti-apoptotic Cell Engineering: a Case Study to Increase the Production Yield of Anti-TNF Alpha Producing Recombinant CHO Cells.

    PubMed

    Gulce Iz, Sultan; Inevi, Muge Anil; Metiner, Pelin Saglam; Tamis, Duygu Ayyildiz; Kisbet, Nazli

    2017-07-06

    Recent developments in medical biotechnology have facilitated to enhance the production of monoclonal antibodies (mAbs) and recombinant proteins in mammalian cells. Human mAbs for clinical applications have focused on three areas, particularly cancer, immunological disorders, and infectious diseases. Tumor necrosis factor alpha (TNF-α), which has both proinflammatory and immunoregulatory functions, is an important target in biopharmaceutical industry. In this study, a humanized anti-TNF-α mAb producing stable CHO cell line which produces a biosimilar of Humira (adalimumab) was used. Adalimumab is a fully human anti-TNF mAb among the top-selling mAb products in recent years as a biosimilar. Products from mammalian cell bioprocesses are a derivative of cell viability and metabolism, which is mainly disrupted by cell death in bioreactors. Thus, different strategies are used to increase the product yield. Suppression of apoptosis, also called anti-apoptotic cell engineering, is the most remarkable strategy to enhance lifetime of cells for a longer production period. In fact, using anti-apoptotic cell engineering as a BioDesign approach was inspired by nature; nature gives prolonged life span to some cells like stem cells, tumor cells, and memory B and T cells, and researchers have been using this strategy for different purposes. In this study, as a biomimicry approach, anti-apoptotic cell engineering was used to increase the anti-TNF-α mAb production from the humanized anti-TNF-α mAb producing stable CHO cell line by Bcl-xL anti-apoptotic protein. It was shown that transient transfection of CHO cells by the Bcl-xL anti-apoptotic protein expressing plasmid prolonged the cell survival rate and protected cells from apoptosis. The transient expression of Bcl-xL using CHO cells enhanced the anti-TNF-α production. The production of anti-TNF-α in CHO cells was increased up to 215 mg/L with an increase of 160% after cells were transfected with Bcl-xL expressing plasmid

  2. 'Super-perfect' enzymes: Structural stabilities and activities of recombinant triose phosphate isomerases from Pyrococcus furiosus and Thermococcus onnurineus produced in Escherichia coli.

    PubMed

    Sharma, Prerna; Guptasarma, Purnananda

    2015-05-08

    Triose phosphate isomerases (TIMs) are considered to be 'kinetically perfect' enzymes, limited in their activity only by the rates of diffusion of substrate and product molecules. Most studies conducted thus far have been on mesophile-derived TIMs. Here, we report studies of two extremophile-derived TIMs produced in Escherichia coli: (i) TonTIM, sourced from the genome of the thermophile archaeon, Thermococcus onnurineus, and (ii) PfuTIM, sourced from the genome of the hyperthermophile archaeon, Pyrococcus furiosus (PfuTIM). Although these enzymes are presumed to have evolved to function optimally at temperatures close to the boiling point of water, we find that TonTIM and PfuTIM display second-order rate-constants of activity (k(cat)/K(m) values) comparable to mesophile-derived TIMs, at 25 °C. At 90 °C, TonTIM and PfuTIM reach maximum velocities of reaction of ∼ 10(6)-10(7) μmol/s/mg, and display k(cat)/K(m) values in the range of ∼ 10(10)-10(11) M(-1) s(-1), which are three orders of magnitude higher than those reported for mesophile TIMs. Further, the two enzymes display no signs of having undergone any structural unfolding at 90 °C. Such enzymes could thus probably be called 'super-perfect' enzymes.

  3. Overexpression of Protein Disulfide Isomerase DsbC Stabilizes Multiple-Disulfide-Bonded Recombinant Protein Produced and Transported to the Periplasm in Escherichia coli

    PubMed Central

    Kurokawa, Yoichi; Yanagi, Hideki; Yura, Takashi

    2000-01-01

    Dsb proteins (DsbA, DsbB, DsbC, and DsbD) catalyze formation and isomerization of protein disulfide bonds in the periplasm of Escherichia coli. By using a set of Dsb coexpression plasmids constructed recently, we analyzed the effects of Dsb overexpression on production of horseradish peroxidase (HRP) isozyme C that contains complex disulfide bonds and tends to aggregate when produced in E. coli. When transported to the periplasm, HRP was unstable but was markedly stabilized upon simultaneous overexpression of the set of Dsb proteins (DsbABCD). Whereas total HRP production increased severalfold upon overexpression of at least disulfide-bonded isomerase DsbC, maximum transport of HRP to the periplasm seemed to require overexpression of all DsbABCD proteins, suggesting that excess Dsb proteins exert synergistic effects in assisting folding and transport of HRP. Periplasmic production of HRP also increased when calcium, thought to play an essential role in folding of nascent HRP polypeptide, was added to the medium with or without Dsb overexpression. These results suggest that Dsb proteins and calcium play distinct roles in periplasmic production of HRP, presumably through facilitating correct folding. The present Dsb expression plasmids should be useful in assessing and dissecting periplasmic production of proteins that contain multiple disulfide bonds in E. coli. PMID:10966415

  4. Social evolution theory for microorganisms.

    PubMed

    West, Stuart A; Griffin, Ashleigh S; Gardner, Andy; Diggle, Stephen P

    2006-08-01

    Microorganisms communicate and cooperate to perform a wide range of multicellular behaviours, such as dispersal, nutrient acquisition, biofilm formation and quorum sensing. Microbiologists are rapidly gaining a greater understanding of the molecular mechanisms involved in these behaviours, and the underlying genetic regulation. Such behaviours are also interesting from the perspective of social evolution - why do microorganisms engage in these behaviours given that cooperative individuals can be exploited by selfish cheaters, who gain the benefit of cooperation without paying their share of the cost? There is great potential for interdisciplinary research in this fledgling field of sociomicrobiology, but a limiting factor is the lack of effective communication of social evolution theory to microbiologists. Here, we provide a conceptual overview of the different mechanisms through which cooperative behaviours can be stabilized, emphasizing the aspects most relevant to microorganisms, the novel problems that microorganisms pose and the new insights that can be gained from applying evolutionary theory to microorganisms.

  5. Spectrum Recombination.

    ERIC Educational Resources Information Center

    Greenslade, Thomas B., Jr.

    1984-01-01

    Describes several methods of executing lecture demonstrations involving the recombination of the spectrum. Groups the techniques into two general classes: bringing selected portions of the spectrum together using lenses or mirrors and blurring the colors by rapid movement or foreshortening. (JM)

  6. Spectrum Recombination.

    ERIC Educational Resources Information Center

    Greenslade, Thomas B., Jr.

    1984-01-01

    Describes several methods of executing lecture demonstrations involving the recombination of the spectrum. Groups the techniques into two general classes: bringing selected portions of the spectrum together using lenses or mirrors and blurring the colors by rapid movement or foreshortening. (JM)

  7. [Bio-active substances derived from marine microorganisms].

    PubMed

    Liu, Quanyong; Hu, Jiangchun; Xue, Delin; Ma, Chengxin; Wang, Shujin

    2002-07-01

    Marine microorganisms, which are taxonomically diverse and genetically special, have powerful potential in producing novel bio-active substances. This article summarized research progress in this respect. The results showed that marine bacteria which are main marine microorganism flora can produce rich kinds of bio-active substances and that even though marine actinomycetes and marine fungi are not as many as marine bacteria in species and quantity, they should be paid no less attention about their bio-active substances. Besides, present research are limited to those marine microorganisms which are easily cultured. One of the future research trends will be focused on bio-active substances derived from non-culturable marine microorganisms.

  8. 40 CFR 725.420 - Recipient microorganisms.

    Code of Federal Regulations, 2013 CFR

    2013-07-01

    ... 40 Protection of Environment 32 2013-07-01 2013-07-01 false Recipient microorganisms. 725.420... CONTROL ACT REPORTING REQUIREMENTS AND REVIEW PROCESSES FOR MICROORGANISMS General Exemptions for New Microorganisms § 725.420 Recipient microorganisms. The following recipient microorganisms are eligible for either...

  9. 40 CFR 725.420 - Recipient microorganisms.

    Code of Federal Regulations, 2014 CFR

    2014-07-01

    ... 40 Protection of Environment 31 2014-07-01 2014-07-01 false Recipient microorganisms. 725.420... CONTROL ACT REPORTING REQUIREMENTS AND REVIEW PROCESSES FOR MICROORGANISMS General Exemptions for New Microorganisms § 725.420 Recipient microorganisms. The following recipient microorganisms are eligible for either...

  10. 40 CFR 725.420 - Recipient microorganisms.

    Code of Federal Regulations, 2012 CFR

    2012-07-01

    ... 40 Protection of Environment 32 2012-07-01 2012-07-01 false Recipient microorganisms. 725.420... CONTROL ACT REPORTING REQUIREMENTS AND REVIEW PROCESSES FOR MICROORGANISMS General Exemptions for New Microorganisms § 725.420 Recipient microorganisms. The following recipient microorganisms are eligible for either...

  11. 40 CFR 725.420 - Recipient microorganisms.

    Code of Federal Regulations, 2010 CFR

    2010-07-01

    ... 40 Protection of Environment 30 2010-07-01 2010-07-01 false Recipient microorganisms. 725.420... CONTROL ACT REPORTING REQUIREMENTS AND REVIEW PROCESSES FOR MICROORGANISMS General Exemptions for New Microorganisms § 725.420 Recipient microorganisms. The following recipient microorganisms are eligible for either...

  12. 40 CFR 725.420 - Recipient microorganisms.

    Code of Federal Regulations, 2011 CFR

    2011-07-01

    ... 40 Protection of Environment 31 2011-07-01 2011-07-01 false Recipient microorganisms. 725.420... CONTROL ACT REPORTING REQUIREMENTS AND REVIEW PROCESSES FOR MICROORGANISMS General Exemptions for New Microorganisms § 725.420 Recipient microorganisms. The following recipient microorganisms are eligible for either...

  13. A Method to Produce and Purify Full-Length Recombinant Alpha Dystroglycan: Analysis of N- and O-Linked Monosaccharide Composition in CHO Cells with or without LARGE Overexpression

    PubMed Central

    Yoon, Jung Hae; Xu, Rui; Martin, Paul

    2013-01-01

    α dystroglycan (αDG) is part of the dystrophin-associated glycoprotein (DAG) complex, a series of cytoskeletal, transmembrane, and membrane-associated proteins that serve to link the extracellular matrix (ECM) surrounding individual skeletal myofibers to the intracellular F-actin cytoskeleton. Glycosylation and ECM protein binding to αDG are regulated by a number of genes that, when defective, give rise to congenital or limb-girdle forms of muscular dystrophy termed dystroglycanopathies. One such dystroglycanopathy gene is LARGE. Here, we describe a method to produce and purify full-length, furin-resistant, recombinant αDG from CHO cells and CHO cells overexpressing LARGE (CHO-LARGE). In addition, we analyze the O- and N-linked monosaccharide composition of such proteins. αDG purified from CHO-LARGE cells had increased molar content of xylose and fucose relative to CHO, while no significant changes were found in N-linked monosaccharides. Glucuronic acid could not be quantified by the methods used. These studies describe a method to produce and purify the milligram amounts of αDG needed for certain biochemical methods, including monosaccharide analysis. Key words: Dystroglycan, muscular dystrophy, xylose, fucose, laminin, LARGE Correspondence: Paul.Martin@nationwidechildrens.org PMID:23390591

  14. [Advances in gene engineering of microorganisms].

    PubMed

    Debabov, V G

    1987-10-01

    A novel branch of national economy--biotechnology is being developed, based on genetic engineering. The construction of strains using the methods of molecular cloning has led to-date to creation of new biotechnological processes. Further advance in biotechnology would be mainly promoted by the possibilities of application of gene engineering to reorganization of industrially important microorganisms. These are bacilli employed for production of vitamins, enzymes, insecticides; streptomycetes--the producers of antibiotics; yeasts applied in bakery industry, in production of fodder proteins; pseudomonads which will be helpful in development of effective biological means for protection of environment, etc. So, vector molecules based on plasmids and phages have been constructed for best-studied representatives of industrial microorganisms, the methods of introduction into the cell of hybrid DNA molecules worked out, the problems of optimization of foreign gene expression being currently solved.

  15. Microorganisms having enhanced tolerance to inhibitors and stress

    SciTech Connect

    Brown, Steven D.; Yang, Shihui

    2014-07-29

    The present invention provides genetically modified strains of microorganisms that display enhanced tolerance to stress and/or inhibitors such as sodium acetate and vanillin. The enhanced tolerance can be achieved by increasing the expression of a protein of the Sm-like superfamily such as a bacterial Hfq protein and a fungal Sm or Lsm protein. Further, the present invention provides methods of producing alcohol from biomass materials by using the genetically modified microorganisms of the present invention.

  16. Recombinant DNA production of spider silk proteins

    PubMed Central

    Tokareva, Olena; Michalczechen-Lacerda, Valquíria A; Rech, Elíbio L; Kaplan, David L

    2013-01-01

    Spider dragline silk is considered to be the toughest biopolymer on Earth due to an extraordinary combination of strength and elasticity. Moreover, silks are biocompatible and biodegradable protein-based materials. Recent advances in genetic engineering make it possible to produce recombinant silks in heterologous hosts, opening up opportunities for large-scale production of recombinant silks for various biomedical and material science applications. We review the current strategies to produce recombinant spider silks. PMID:24119078

  17. Recombinant DNA production of spider silk proteins.

    PubMed

    Tokareva, Olena; Michalczechen-Lacerda, Valquíria A; Rech, Elíbio L; Kaplan, David L

    2013-11-01

    Spider dragline silk is considered to be the toughest biopolymer on Earth due to an extraordinary combination of strength and elasticity. Moreover, silks are biocompatible and biodegradable protein-based materials. Recent advances in genetic engineering make it possible to produce recombinant silks in heterologous hosts, opening up opportunities for large-scale production of recombinant silks for various biomedical and material science applications. We review the current strategies to produce recombinant spider silks.

  18. Adoptive transfer of experimental allergic encephalomyelitis after in vitro treatment with recombinant murine interleukin-12. Preferential expansion of interferon-gamma-producing cells and increased expression of macrophage-associated inducible nitric oxide synthase as immunomodulatory mechanisms.

    PubMed Central

    Waldburger, K. E.; Hastings, R. C.; Schaub, R. G.; Goldman, S. J.; Leonard, J. P.

    1996-01-01

    In an adoptive transfer model of experimental allergic encephalomyelitis, stimulation of lymph node cells with proteolipid protein and recombinant murine interleukin (rmIL)-12 before cell transfer accelerated the onset and exacerbates clinical disease. In vitro stimulation with proteolipid protein in the presence of rmIL-12 was associated with an increase in interferon-gamma-producing cells and a decrease in IL-4-producing cells, indicating a preferential expansion of Th1 effector cells. This was supported by the finding that severe disease with rapid onset could be transferred with as few as 10 x 10(6) rmIL-12-stimulated lymph node cells. Immunohistochemical analysis confirmed that the accelerated onset of disease after in vitro stimulation with rmIL-12 coincided with an acute inflammatory response in the central nervous system. At peak disease, both control and rmIL-12 treatment groups exhibited extensive cellular infiltration with characteristic perivascular cuffing. No notable differences in either the cellular composition or cytokine expression within the lesions were seen between groups. However, the frequency of macrophages that stained positively for inducible nitric oxide synthase was increased in animals challenged with rmIL-12-treated lymph node cells. The results suggest that, in addition to promoting the preferential expansion of interferon-gamma-producing cells by rmIL-12 in vitro, secondary in vivo effects leading to macrophage activation and inducible nitric oxide synthase expression may contribute to the severe and protracted course of central nervous system inflammation in this model. Images Figure 2 PMID:8579100

  19. Recombinant protein expression plasmids optimized for industrial E. coli fermentation and plant systems produce biologically active human insulin-like growth factor-1 in transgenic rice and tobacco plants.

    PubMed

    Panahi, Mitra; Alli, Zaman; Cheng, Xiongying; Belbaraka, Loubaba; Belgoudi, Jaafar; Sardana, Ravinder; Phipps, Jenny; Altosaar, Illimar

    2004-06-01

    Human insulin-like growth factor-1 (hIGF-1) is a growth factor with clinical significance in medicine. The therapeutic potential of recombinant hIGF-1 (rthIGF-1) stems from the fact that hIGF-1 resembles insulin in many aspects of physiology. The expression of hIGF-1 in transgenic tobacco and rice plants using different expression cassettes is reported here. In the present study, two coding sequences were tested, one with the original human sequence, but partially optimized for expression in E. coli and the other with a plant-codon-optimized sequence that was expected to give a higher level of expression in plant systems. Three different hIGF-1 recombinant expression constructs were generated. All expression constructs utilized the maize ubiquitin 1 promoter with or without a signal sequence. Analyses conducted using a hIGF-1 specific ELISA kit showed all transgenic plants produced hIGF-1 and the accumulated hIGF-1 increased from the E. coli codon bias to higher levels when the hIGF-1 coding sequence was codon-optimized to match that of the maize zeamatin protein--the most transcribed gene in maize endosperm suspension cells. Further analyses that compared the functionality of the bacterial signal peptide Lam B in plants showed that this leader peptide led to lower expression levels when compared to transgenic plants that did not contain this sequence. This indicated that this expression construct was functional without removal of the bacterial signal sequence. The maize ubiquitin 1 promoter was found to be more active in rice plants than tobacco plants indicating that in this case, there was a class preference that was biased towards a monocot host. Biological analyses conducted using protein extracts from transgenic plants showed that the rthIGF-1 was effective in stimulating the in vitro growth and proliferation of human SH-SY5Y neuroblastoma cells. This indicated that the plant-produced rthIGF-1 was stable and biologically active. As some plants have been

  20. Antibiotic resistant in microorganisms

    USDA-ARS?s Scientific Manuscript database

    Antimicrobial agents are necessary for use in veterinary medicine including the production of food producing animals. Antibiotic use is indicated for the treatment of bacterial target organisms and/or disease for which the antibiotic was developed. However, an unintended consequence of antibiotic ...

  1. Sensor arrays for detecting microorganisms

    NASA Technical Reports Server (NTRS)

    Lewis, Nathan S. (Inventor); Freund, Michael S. (Inventor)

    2000-01-01

    A sensor array for detecting a microorganism comprising first and second sensors electrically connected to an electrical measuring apparatus, wherein the sensors comprise a region of nonconducting organic material and a region of conducting material compositionally that is different than the nonconducting organic material and an electrical path through the regions of nonconducting organic material and the conducting material. A system for identifying microorganisms using the sensor array, a computer and a pattern recognition algorithm, such as a neural net are also disclosed.

  2. [Spreading and mechanisms of antibiotic resistance of microorganisms, producing beta-lactamases. Molecular mechanisms of resistance to beta-lactams of Klebsiella spp. strains, isolated in cases of nosocomial infections].

    PubMed

    Ivanov, D V; Egorov, A M

    2008-01-01

    Antibiotic sensivity of nosocomial Klebsiella spp. strains (n = 212), isolated from patients treated in 30 medical centers of 15 various regions of Russia was investigated. The Klebsiella genus was represented by the following species: Klebsiella pneumoniae ss. pneumoniae--182 (85.8%), Klebsiella pneumoniae ss. ozaenae--1 (0.5%), Klebsiella oxytoca--29 (13.7%) isolates. The most active antibacterial agents against the investigated strains were carbapenems (imipenem and meropenem). Among 3rd generation cephalosporine the lowest MICs were observed for ceftazidime/clavulanic acid (MIC50--0.25 microg/ml, MIC90--64 microg/ml) and cefoperazone/sulbactam (MIC50--16 microg/ml, MIC90--64 microg/ml). Beta-lactamase genes (TEM, SHV, CTX) were detected in 42 Klebsiella pneumoniae ss. pneumoniae strains by PCR. Alone or in various combinations TEM type beta-lactamases have been found in 16 (38.1%) isolates, SHV--in 29 (69%), and CTX--in 27 (64.3%). Combinations of 2 different determinants were detected in 23.8% of the isolates, 3--in 26.2%. There were not isolates producing MBL class B among resistant to carbapenems nosocomial Klebsiella spp. strains.

  3. Textiles for protection against microorganism

    NASA Astrophysics Data System (ADS)

    Sauperl, O.

    2016-04-01

    Concerning micro-organisms such as bacteria, viruses and fungi, there is a huge progress in the development of textile materials and procedures which should effectively protect against these various pathogens. In this sense there is especially problematic hospital environment, where it is necessary to take into account properly designed textile material which, when good selected and composed, act as a good barrier against transfer of micro-organisms through material mainly in its wet state. Respect to this it is necessary to be familiar with the rules regarding selection of the input material, the choice of proper yarn construction, the choice of the proper weaving mode, the rules regarding selection of antimicrobial-active compound suitable for (eco-friendly) treatment, and the choice of the most appropriate test method by which it is possible objectively to conclude on the reduction of selected microorganism. As is well known, fabrics are three-dimensional structures with void and non-void areas. Therefore, the physical-chemical properties of the textile material/fabric, the surface characteristics together with the shape of microorganism, and the carriers' characteristics contribute to control the transfer of microorganism through textile material. Therefore, careful planning of textile materials and treatment procedure with the compound which is able to reduce micro-organism satisfactory is particularly important, especially due to the fact that in hospital environment population with impaired immune system is mainly presented.

  4. A novel NADH-dependent carbonyl reductase with unusual stereoselectivity for (R)-specific reduction from an (S)-1-phenyl-1,2-ethanediol-producing micro-organism: purification and characterization.

    PubMed

    Nie, Y; Xu, Y; Yang, M; Mu, X-Q

    2007-05-01

    To purify and characterize the (R)-specific carbonyl reductase from Candida parapsilosis; to compare the enzyme with other stereospecific oxidoreductases; and to develop an available procedure producing optically active (R)-1-phenyl-1,2-ethanediol (PED). An (R)-specific carbonyl reductase was found and purified from C. parapsilosis through four steps, including blue-sepharose affinity chromatography. The relative molecular mass of the enzyme was estimated to be 35 kDa on gel-filtration chromatography and 37.5 kDa on Sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The purified enzyme catalysed the reduction of various ketones, including alkyl and aromatic ketones, and was specific to short-chain and medium-chain alkyl ketones. The enzyme activity was inhibited by divalent ion of CuSO(4) and FeSO(4), whereas zincum ion stimulated its activity. For catalysing reduction, the enzyme performed maximum activity at pH 6.0 and the optimum temperature was 45 degrees C. The carbonyl reductase catalysed asymmetric reduction of beta-hydroxyacetophenone to the corresponding (R)-PED with the optical purity of 100% enantiomeric excess (e.e.). By analysing its partial amino acid sequences, the enzyme was proposed to be a novel stereospecific carbonyl reductase. The purified carbonyl reductase showed unusual stereospecificity and catalysed the NADH-dependent reduction of beta-hydroxyacetophenone to (R)-PED. The enzyme was different from other stereoselective oxidoreductases in catalytic properties. The discovery of (R)-specific oxidoreductase exhibiting unusual stereospecificity towards hydroxyl ketone is valuable for the synthesis of both enantiomers of useful chiral alcohols, and provides research basis for the achievement of profound knowledge on the relationship between structure and catalytic function of (R)-specific enzymes, which is meaningful for the alteration of stereospecificity by molecular methods to obtain the enzymes with desired stereospecificity.

  5. Screening For Alcohol-Producing Microbes

    NASA Technical Reports Server (NTRS)

    Schubert, Wayne W.

    1988-01-01

    Dye reaction rapidly identifies alcohol-producing microbial colonies. Method visually detects alcohol-producing micro-organisms, and distinguishes them from other microbial colonies that do not produce alcohol. Method useful for screening mixed microbial populations in environmental samples.

  6. Engineered biosealant strains producing inorganic and organic biopolymers.

    PubMed

    Bergdale, Terran E; Pinkelman, Rebecca J; Hughes, Stephen R; Zambelli, Barbara; Ciurli, Stefano; Bang, Sookie S

    2012-10-31

    Microbiologically induced calcium carbonate precipitation (MICCP) is a naturally occurring biological process that has shown its potential in remediation of a wide range of structural damages including concrete cracks. In this study, genetically engineered microorganisms, capable of producing extracellular polymeric substances (EPSs) as well as inducing MICCP, were developed based on the assumption that the complex of inorganic CaCO(3) and organic EPS would provide a stronger matrix than MICCP alone as biosealant. In order to develop a recombinant biosealant microorganism, the entire Sporosarcina pasteurii urease gene sequences including ureA, ureB, ureC, ureD, ureE, ureF, and ureG from plasmid pBU11 were sub-cloned into the shuttle vector, pUCP18. The newly constructed plasmid, pUBU1, was transformed into two Pseudomonas aeruginosa strains, 8821 and PAO1, to develop recombinants capable of inducing calcite precipitation in addition to their own ability to produce EPS. Nickel-dependent urease activities were expressed from the recombinant P. aeruginosa 8821 (pUBU1) and P. aeruginosa PAO1 (pUBU1), at 99.4% and 60.9% of the S. pasteurii urease activity, respectively, in a medium containing 2mM NiCl(2). No urease activities were detected from the wild type P. aeruginosa 8821 and P. aeruginosa PAO1 under the same growth conditions. Recombinant Pseudomonas strains induced CaCO(3) precipitation at a comparable rate as S. pasteurii and scanning electron microscopy evidenced the complex of CaCO(3) crystals and EPS layers surrounding the cells. The engineered strains produced in this study are expected to serve as a valuable reference to future biosealants that could be applied in the environment. However, the pathogenic potential of P. aeruginosa, used here only as a model system to show the proof of principle, prevents the use of this recombinant organism as a biosealant. In practical applications, other recombinant organisms should be used. Copyright © 2012 Elsevier B

  7. Biomining: metal recovery from ores with microorganisms.

    PubMed

    Schippers, Axel; Hedrich, Sabrina; Vasters, Jürgen; Drobe, Malte; Sand, Wolfgang; Willscher, Sabine

    2014-01-01

    Biomining is an increasingly applied biotechnological procedure for processing of ores in the mining industry (biohydrometallurgy). Nowadays the production of copper from low-grade ores is the most important industrial application and a significant part of world copper production already originates from heap or dump/stockpile bioleaching. Conceptual differences exist between the industrial processes of bioleaching and biooxidation. Bioleaching is a conversion of an insoluble valuable metal into a soluble form by means of microorganisms. In biooxidation, on the other hand, gold is predominantly unlocked from refractory ores in large-scale stirred-tank biooxidation arrangements for further processing steps. In addition to copper and gold production, biomining is also used to produce cobalt, nickel, zinc, and uranium. Up to now, biomining has merely been used as a procedure in the processing of sulfide ores and uranium ore, but laboratory and pilot procedures already exist for the processing of silicate and oxide ores (e.g., laterites), for leaching of processing residues or mine waste dumps (mine tailings), as well as for the extraction of metals from industrial residues and waste (recycling). This chapter estimates the world production of copper, gold, and other metals by means of biomining and chemical leaching (bio-/hydrometallurgy) compared with metal production by pyrometallurgical procedures, and describes new developments in biomining. In addition, an overview is given about metal sulfide oxidizing microorganisms, fundamentals of biomining including bioleaching mechanisms and interface processes, as well as anaerobic bioleaching and bioleaching with heterotrophic microorganisms.

  8. Recombinant Lactococcus lactis fails to secrete bovine chymosine

    PubMed Central

    Luerce, Tessália Diniz; Azevedo, Marcela Santiago Pacheco; LeBlanc, Jean Guy; Azevedo, Vasco; Miyoshi, Anderson; Pontes, Daniela Santos

    2014-01-01

    Bovine chymosin is an important milk-clotting agent used in the manufacturing of cheeses. Currently, the production of recombinant proteins by genetically modified organisms is widespread, leading to greatly reduced costs. Lactococcus (L.) lactis, the model lactic acid bacterium, was considered a good candidate for heterologous chymosin production for the following reasons: (1) it is considered to be a GRAS (generally regarded as safe) microorganism, (2) only one protease is present on its surface, (3) it can secrete proteins of different sizes, and (4) it allows for the direct production of protein in fermented food products. Thus, three genetically modified L. lactis strains were constructed to produce and target the three different forms of bovine chymosin, prochymosin B, chymosin A and chymosin B to the extracellular medium. Although all three proteins were stably produced in L. lactis, none of the forms were detected in the extracellular medium or showed clotting activity in milk. Our hypothesis is that this secretion deficiency and lack of clotting activity can be explained by the recombinant protein being attached to the cell envelope. Thus, the development of other strategies is necessary to achieve both production and targeting of chymosin in L. lactis, which could facilitate the downstream processing and recovery of this industrially important protein. PMID:25482140

  9. Recombinant Lactococcus lactis fails to secrete bovine chymosine.

    PubMed

    Luerce, Tessália Diniz; Azevedo, Marcela Santiago Pacheco; LeBlanc, Jean Guy; Azevedo, Vasco; Miyoshi, Anderson; Pontes, Daniela Santos

    2014-01-01

    Bovine chymosin is an important milk-clotting agent used in the manufacturing of cheeses. Currently, the production of recombinant proteins by genetically modified organisms is widespread, leading to greatly reduced costs. Lactococcus (L.) lactis, the model lactic acid bacterium, was considered a good candidate for heterologous chymosin production for the following reasons: (1) it is considered to be a GRAS (generally regarded as safe) microorganism, (2) only one protease is present on its surface, (3) it can secrete proteins of different sizes, and (4) it allows for the direct production of protein in fermented food products. Thus, three genetically modified L. lactis strains were constructed to produce and target the three different forms of bovine chymosin, prochymosin B, chymosin A and chymosin B to the extracellular medium. Although all three proteins were stably produced in L. lactis, none of the forms were detected in the extracellular medium or showed clotting activity in milk. Our hypothesis is that this secretion deficiency and lack of clotting activity can be explained by the recombinant protein being attached to the cell envelope. Thus, the development of other strategies is necessary to achieve both production and targeting of chymosin in L. lactis, which could facilitate the downstream processing and recovery of this industrially important protein.

  10. Ethanol production by recombinant hosts

    DOEpatents

    Ingram, Lonnie O.; Beall, David S.; Burchhardt, Gerhard F. H.; Guimaraes, Walter V.; Ohta, Kazuyoshi; Wood, Brent E.; Shanmugam, Keelnatham T.

    1995-01-01

    Novel plasmids comprising genes which code for the alcohol dehydrogenase and pyruvate decarboxylase are described. Also described are recombinant hosts which have been transformed with genes coding for alcohol dehydrogenase and pyruvate. By virtue of their transformation with these genes, the recombinant hosts are capable of producing significant amounts of ethanol as a fermentation product. Also disclosed are methods for increasing the growth of recombinant hosts and methods for reducing the accumulation of undesirable metabolic products in the growth medium of these hosts. Also disclosed are recombinant host capable of producing significant amounts of ethanol as a fermentation product of oligosaccharides and plasmids comprising genes encoding polysaccharases, in addition to the genes described above which code for the alcohol dehydrogenase and pyruvate decarboxylase. Further, methods are described for producing ethanol from oligomeric feedstock using the recombinant hosts described above. Also provided is a method for enhancing the production of functional proteins in a recombinant host comprising overexpressing an adhB gene in the host. Further provided are process designs for fermenting oligosaccharide-containing biomass to ethanol.

  11. Ethanol production by recombinant hosts

    DOEpatents

    Fowler, David E.; Horton, Philip G.; Ben-Bassat, Arie

    1996-01-01

    Novel plasmids comprising genes which code for the alcohol dehydrogenase and pyruvate decarboxylase are described. Also described are recombinant hosts which have been transformed with genes coding for alcohol dehydrogenase and pyruvate. By virtue of their transformation with these genes, the recombinant hosts are capable of producing significant amounts of ethanol as a fermentation product. Also disclosed are methods for increasing the growth of recombinant hosts and methods for reducing the accumulation of undesirable metabolic products in the growth medium of these hosts. Also disclosed are recombinant host capable of producing significant amounts of ethanol as a fermentation product of oligosaccharides and plasmids comprising genes encoding polysaccharases, in addition to the genes described above which code for the alcohol dehydrogenase and pyruvate decarboxylase. Further, methods are described for producing ethanol from oligomeric feedstock using the recombinant hosts described above. Also provided is a method for enhancing the production of functional proteins in a recombinant host comprising overexpressing an adhB gene in the host. Further provided are process designs for fermenting oligosaccharide-containing biomass to ethanol.

  12. Screening of biosurfactants from cloud microorganisms

    NASA Astrophysics Data System (ADS)

    Sancelme, Martine; Canet, Isabelle; Traikia, Mounir; Uhliarikova, Yveta; Capek, Peter; Matulova, Maria; Delort, Anne-Marie; Amato, Pierre

    2015-04-01

    The formation of cloud droplets from aerosol particles in the atmosphere is still not well understood and a main source of uncertainties in the climate budget today. One of the principal parameters in these processes is the surface tension of atmospheric particles, which can be strongly affected by trace compounds called surfactants. Within a project devoted to bring information on atmospheric surfactants and their effects on cloud droplet formation, we focused on surfactants produced by microorganisms present in atmospheric waters. From our unique collection of microorganisms, isolated from cloud water collected at the Puy-de-Dôme (France),1 we undertook a screening of this bank for biosurfactant producers. After extraction of the supernatants of the pure cultures, surface tension of crude extracts was determined by the hanging drop technique. Results showed that a wide variety of microorganisms are able to produce biosurfactants, some of them exhibiting strong surfactant properties as the resulting tension surface decreases to values less then 35 mN.m-1. Preliminary analytical characterization of biosurfactants, obtained after isolation from overproducing cultures of Rhodococcus sp. and Pseudomonas sp., allowed us to identify them as belonging to two main classes, namely glycolipids and glycopeptides. 1. Vaïtilingom, M.; Attard, E.; Gaiani, N.; Sancelme, M.; Deguillaume, L.; Flossmann, A. I.; Amato, P.; Delort, A. M. Long-term features of cloud microbiology at the puy de Dôme (France). Atmos. Environ. 2012, 56, 88-100. Acknowledgements: This work is supported by the French-USA ANR SONATA program and the French-Slovakia programs Stefanik and CNRS exchange.

  13. Antigenic and immunogenic properties of recombinant hemagglutinin proteins from H1N1 A/Brisbane/59/07 and B/Florida/04/06 when produced in various protein expression systems

    PubMed Central

    Santiago, Felix W.; Emo, Kris Lambert; Fitzgerald, Theresa; Treanor, John J.

    2012-01-01

    Antibodies directed against the influenza hemagglutinin (HA) protein largely mediate virus neutralization and confer protection against infection. Consequently, many studies and assays of influenza vaccines are focused on HA-specific immune responses. Recombinant HA (rHA) proteins can be produced in a number of protein expression and cell culture systems. These range from baculovirus infection of insect cell cultures, to transient transfection of plants, to stably transfected human cell lines. Furthermore, the rHA proteins may contain genetic modifications, such as histidine tags or trimerization domains, intended to ease purification or enhance protein stability. However, no systematic study of these different forms of the HA protein have been conducted. It is not clear which, if any, of these different protein expression systems or structural modifications improve or diminish the biological behavior of the proteins as immunogens or antigens in immune assays. Therefore we set out to perform systematic evaluation of rHA produced in different proteins expression systems and with varied modifications. Five rHA proteins based on recent strains of seasonal influenza A and five based on influenza B HA were kindly provided by the Biodefense and Emerging Infections Reagent repository (BEIR). These proteins were evaluated in a combination of biochemical and structural assays, in vitro humoral and cellular immune assays, and in an animal vaccination model. Marked differences in the behavior of the individual proteins was evident suggesting that they are not equal when being used to detect an immune response. They were, nevertheless, similar at eliciting neutralizing antibody responses. PMID:22609035

  14. Marine microorganisms as potential biofactories for synthesis of metallic nanoparticles.

    PubMed

    Manivasagan, Panchanathan; Nam, Seung Yun; Oh, Junghwan

    2016-11-01

    The use of marine microorganisms as potential biofactories for green synthesis of metallic nanoparticles is a relatively new field of research with considerable prospects. This method is eco-friendly, time saving, and inexpensive and can be easily scaled up for large-scale synthesis. The increasing need to develop simple, nontoxic, clean, and environmentally safe production methods for nanoparticles and to decrease environmental impact, minimize waste, and increase energy productivity has become important in this field. Marine microorganisms are tiny organisms that live in marine ecosystems and account for >98% of biomass of the world's ocean. Marine microorganisms synthesize metallic nanoparticles either intracellularly or extracellularly. Marine microbially-produced metallic nanoparticles have received considerable attention in recent years because of their expected impact on various applications such as medicine, energy, electronic, and space industries. The present review discusses marine microorganisms as potential biofactories for the green synthesis of metallic nanoparticles and their potential applications.

  15. Effects of beneficial microorganisms on lowland rice development.

    PubMed

    Nascente, Adriano Stephan; de Filippi, Marta Cristina Corsi; Lanna, Anna Cristina; de Sousa, Thatyane Pereira; de Souza, Alan Carlos Alves; da Silva Lobo, Valácia Lemes; da Silva, Gisele Barata

    2017-09-19

    Microorganisms can promote plant growth by increasing phytomass production, nutrient uptake, photosynthesis rates, and grain yield, which can result in higher profits for farmers. However, there is limited information available about the physiological characteristics of lowland rice after treatment with beneficial microorganisms in the tropical region. This study aimed to determine the effects of different beneficial microorganisms and various application forms on phytomass production, gas exchange, and nutrient contents in the lowland rice cultivar 'BRS Catiana' in a tropical region. The experiment was performed under greenhouse conditions utilizing a completely randomized design and a 7 × 3 + 1 factorial scheme with four replications. The treatments consisted of seven microorganisms, including the rhizobacterial isolates BRM 32113, BRM 32111, BRM 32114, BRM 32112, BRM 32109, and BRM 32110 and Trichoderma asperellum pooled isolates UFRA-06, UFRA-09, UFRA-12, and UFRA-52, which were applied using three different methods (microbiolized seed, microbiolized seed + soil drenched with a microorganism suspension at 7 and 15 days after sowing (DAS), and microbiolized seed + plant spraying with a microorganism suspension at 7 and 15 DAS) with a control (water). The use of microorganisms can provide numerous benefits for rice in terms of crop growth and development. The microorganism types and methods of application positively and differentially affected the physiological characteristics evaluated in the experimental lowland rice plants. Notably, the plants treated with the bioagent BRM 32109 on the seeds and on seeds + soil produced plants with the highest dry matter biomass, gas exchange rate, and N, P, Fe, and Mg uptake. Therefore, our findings indicate strong potential for the use of microorganisms in lowland rice cultivation systems in tropical regions. Currently, an additional field experiment is in its second year to validate the beneficial result reported

  16. Construction of a genetically engineered microorganism with high tolerance to arsenite and strong arsenite oxidative ability.

    PubMed

    Yang, Chunyan; Xu, Lin; Yan, Limin; Xu, Yanhua

    2010-01-01

    Genetically engineered microorganisms (GEMs) have shown great potential for use in environmental bioremediation. In this study, the TTHB128 and TTHB127 genes, which encode the small and large subunits of arsentie oxidase in Thermus thermophilus HB8, respectively, were cloned into the broad-host-range vector pBBR1MCS-5 to produce the recombinant plasmid, TTHB127-pBBR1MCS-5-TTHB128. This resulted in successful construction of a GEM with high tolerance to arsenite and strong arsenite oxidative ability. Culture of the GEM in media containing arsenite for 28 h resulted in 87.6% of the arsenite being oxidized. Overall, the oxidative ability of the GEM was much stronger than that of the wild type host strain. Gentamicin was necessary to maintain the stability of the recombinant plasmid, TTHB127-pBBR1MCS-5-TTHB128, in the GEM. The oxidative ability of the GEM remained unchanged when it was grown in medium containing gentamicin (60 mg/L) for 30 growth cycles, after which its activity gradually decreased.

  17. Industrial PE-2 strain of Saccharomyces cerevisiae: from alcoholic fermentation to the production of recombinant proteins.

    PubMed

    Soares-Costa, Andrea; Nakayama, Darlan Gonçalves; Andrade, Letícia de Freitas; Catelli, Lucas Ferioli; Bassi, Ana Paula Guarnieri; Ceccato-Antonini, Sandra Regina; Henrique-Silva, Flavio

    2014-01-25

    Saccharomyces cerevisiae is the most important microorganism used in the ethanol fermentation process. The PE-2 strain of this yeast is widely used to produce alcohol in Brazil due to its high fermentation capacity. The aim of the present study was to develop an expression system for recombinant proteins using the industrial PE-2 strain of S. cerevisiae during the alcoholic fermentation process. The protein chosen as a model for this system was CaneCPI-1, a cysteine peptidase inhibitor. A plasmid containing the CaneCPI-1 gene was constructed and yeast cells were transformed with the pYADE4_CaneCPI-1 construct. To evaluate the effect on fermentation ability, the transformed strain was used in the fermentation process with cell recycling. During the nine-hour fermentative cycles the transformed strain did not have its viability and fermentation ability affected. In the last cycle, when the fermentation lasted longer, the protein was expressed probably at the expense of ethanol once the sugars were exhausted. The recombinant protein was expressed in yeast cells, purified and submitted to assays of activity that demonstrated its functionality. Thus, the industrial PE-2 strain of S. cerevisiae can be used as a viable system for protein expression and to produce alcohol simultaneously. The findings of the present study demonstrate the possibility of producing recombinant proteins with biotechnological applications during the ethanol fermentation process.

  18. Why are some microorganisms boring?

    PubMed

    Cockell, Charles S; Herrera, Aude

    2008-03-01

    Microorganisms from diverse environments actively bore into rocks, contributing significantly to rock weathering. Carbonates are the most common substrate into which they bore, although there are also reports of microbial borings into volcanic glass. One of the most intriguing questions in microbial evolutionary biology is why some microorganisms bore. A variety of possible selection pressures, including nutrient acquisition, protection from UV radiation and predatory grazing could promote boring. None of these pressures is mutually exclusive and many of them could have acted in concert with varying strengths in different environments to favour the development of microorganisms that bore. We suggest that microbial boring might have begun in some environments as a mechanism against entombment by mineralization.

  19. PCB breakdown by anaerobic microorganisms

    SciTech Connect

    Not Available

    1989-03-01

    Recently, altered PCB cogener distribution patterns observed in anaerobic sediment samples from the upper Hudson River are being attributed to biologically mediated reductive dechlorination. The authors report their successful demonstration of biologically mediated reductive dechlorination of an Aroclor mixture. In their investigation, they assessed the ability of microorganisms from PCB-contaminated Hudson River sediments (60-562 ppm PCBs) to dechlorinate Aroclor 1242 under anaerobic conditions by eluting microorganisms from the PCB- contaminated sediments and transferring them to a slurry of reduced anaerobic mineral medium and PCB-free sediments in tightly stoppered bottles. They observed dechlorination to be the most rapid at the highest PCB concentration tried by them.

  20. [Producing of molecular hydrogen by association of sporulating microorganisms].

    PubMed

    Matveeva, N A; Levishko, A S; Pritula, I R; Tashireva, A A; Rokitko, P V; Tashirev, A B

    2011-01-01

    Technologically promising microbe association, consisting of aerobic and anaerobic sporulating bacteria has been isolated. The association synthesizes molecular hydrogen during fermentation of potato and starch. The association was isolated from soil, pasteurized on the boiling water bath. The association destroys potato during 5-7 days with a decrease of mass up to 17.4 times and synthesizes gas consisting of 60% of H2.

  1. Comparative Analyses of Exoproteinases Produced by Three Phytopathogenic Microorganisms

    PubMed Central

    Valueva, Tatiana A.; Kudryavtseva, Natalia N.; Sof'in, Alexis V.; Revina, Tatiana A.; Gvozdeva, Ekaterina L.; Ievleva, Elena V.

    2011-01-01

    Proteinases secreted by the oomycete Phytophthora infestans (Mont.) de Bary, Rhizoctonia solani, and Fusarium culmorum belonging to different families of fungi have been studied to determine if the exoenzyme secretion depends on the environmental conditions and the phylogenetic position of the pathogen. The substrate specificity of the extracellular proteinases of F. culmorum, R. solani, and P. infestans and their sensitivity to the action of synthetic and protein inhibitors suggest that they contain trypsin-like and subtilisin-like enzymes regardless of culture medium composition. The relation of trypsin-like and subtilisin-like enzymes is dependent on the culture medium composition, especially on the form of nitrogen nutrition, particularly in the case of the exoenzymes secreted by R. solani. Phylogenetic analyses have shown that the exoproteinase set of ascomycetes and oomycetes has more similarities than basidiomycetes although they are more distant relatives. Our data suggests that the multiple proteinases secreted by pathogenic fungi could play different roles in pathogenesis, increasing the adaptability and host range, or could have different functions in survival in various ecological habitats outside the host. PMID:22567343

  2. Cells and methods for producing fatty alcohols

    DOEpatents

    Pfleger, Brian F.; Youngquist, Tyler J.

    2017-07-18

    Recombinant cells and methods for improved yield of fatty alcohols. The recombinant cells harbor a recombinant thioesterase gene, a recombinant acyl-CoA synthetase gene, and a recombinant acyl-CoA reductase gene. In addition, a gene product from one or more of an acyl-CoA dehydrogenase gene, an enoyl-CoA hydratase gene, a 3-hydroxyacyl-CoA dehydrogenase gene, and a 3-ketoacyl-CoA thiolase gene in the recombinant cells is functionally deleted. Culturing the recombinant cells produces fatty alcohols at high yields.

  3. In vitro stimulation of Balb/c and C57 BL/6 splenocytes by a recombinantly produced banana lectin isoform results in both a proliferation of T cells and an increased secretion of interferon-gamma.

    PubMed

    Stojanović, Marijana M; Zivković, Irena P; Petrusić, Vladimir Z; Kosec, Dusko J; Dimitrijević, Rajna D; Jankov, Ratko M; Dimitrijević, Ljiljana A; Gavrović-Jankulović, Marija D

    2010-01-01

    Lectins are widely used in many types of assay but some lectins such as banana lectin (BanLec) are recognised as potent immunostimulators. Although BanLec's structure and binding characteristics are now familiar, its immunostimulatory potential has not yet been fully explored. The synthesis by recombinant technology of a BanLec isoform (rBanLec) whose binding properties are similar to its natural counterpart has made it possible to overcome the twin problems of natural BanLec's microheterogeneity and low availability. This study's aim is to explore the immunostimulatory potential of rBanLec in the murine model. Analyses of the responses of Balb/c- and C57 BL/6-originated splenocytes to in vitro rBanLec stimulation were performed to examine the dependency of rBanLec's immunostimulatory potential upon the splenocytes' genetic background. It is shown that the responses of Balb/c- and C57 BL/6-originated splenocytes to rBanLec stimulation differ both qualitatively and in intensity. The hallmarks of the induced responses are T lymphocyte proliferation and intensive interferon-gamma secretion. Both phenomena are more marked in Balb/c-originated cultures; Balb/c-originated lymphocytes produce interleukin (IL)-4 and IL-10 following rBanLec stimulation. Our results demonstrate that any responses to rBanLec stimulation are highly dependent upon genetic background; they suggest that genetic background must be an important consideration in any further investigations using animal models or when exploring rBanLec's potential human applications.

  4. Testing for recombinant erythropoietin.

    PubMed

    Delanghe, Joris R; Bollen, Mathieu; Beullens, Monique

    2008-03-01

    Erythropoietin (Epo) is a glycoprotein hormone that promotes the production of red blood cells. Recombinant human Epo (rhEpo) is illicitly used to improve performance in endurance sports. Doping in sports is discouraged by the screening of athletes for rhEpo. Both direct tests (indicating the presence of exogeneous Epo isoforms) and indirect tests (indicating hematological changes induced by exogenous Epo administration) can be used for Epo detection. At present, the test adopted by the World Anti Doping Agency is based on a combination of isoelectric focusing and double immunoblotting, and distinguishes between endogenous and rhEpo. However, the adopted monoclonal anti-Epo antibodies are not monospecific. Therefore, the test can occasionally lead to the false-positive detection of rhEpo (epoetin-beta) in post-exercise, protein-rich urine, or in case of contamination of the sample with microorganisms. An improved preanalytical care may counteract a lot of these problems. Adaptation of the criteria may be helpful to further refine direct Epo testing. Indirect tests have the disadvantage that they require blood instead of urine samples, but they can be applied to detect a broader range of performance improving techniques which are illicitly used in sports.

  5. Quantity analysis of micro-organisms in bottled water

    NASA Astrophysics Data System (ADS)

    Li, Juan; Li, Xiangyong

    2008-12-01

    Water is necessary to human being and all kinds of animals and plants. In recently years, Bottled Water become the main drinking water whatever for families or for institutions. But most of them have no conception of the water's safety or quality. To have conceptions of the count and distributing of the microorganisms in bucket pure water, we use fluorescent microscope counting stained with SYBR Green I to research the microorganisms (including virus) quantity in Bottled Water for six samples produced in different place. Analyzing shows that the quantity of the microorganisms in these water are different. Some up to 11.912×106 virus/ m L. The quality of Bottled Water needs to be improved. And the quantity of microorganisms in the water is different with different ways to keep the water. At the same time, it shows that fluorescent microscope counting stained with SYBR Green I method is simple and high sensitive to such low microorganisms quantity water sample. It can be used in the microorganisms dynamic quantity research in drinking water.

  6. New therapeutic approaches by using microorganism-derived compounds.

    PubMed

    Amedei, A; D'Elios, M M

    2012-01-01

    The role of natural products as a source for remedies has been recognized since ancient times. Despite major scientific and technological progress in combinatorial chemistry, drugs derived from natural product still make an enormous contribution to drug discovery today. Nature is an attractive source of new therapeutic candidate compounds since a tremendous chemical diversity is found in millions of species of plants, animals, marine organisms and microorganisms. Microorganisms such as bacteria and fungi have been invaluable to discover drugs and lead compounds. These microorganisms produce a large variety of antimicrobial agents which have evolved to give their hosts an advantage over their competitors in the microbiological world. The screening of microorganisms became highly popular after the discovery of penicillin but in recent years the list of antibacterial agents (bacteria- or fungi-derived) has increased considerably with the arrival of cephalosporins, tetracyclines, aminoglycosides, rifamycins, and chloramphenicol. Although most of the drugs derived from microorganisms are used in antibacterial therapy, some microbial metabolites have provided lead compounds in other fields of medicine. For example: the fungal metabolite lovastatin, which was the lead compound for a series of drugs that lower cholesterol levels, the ciclosporin (fungal metabolite) currently used to suppress the immune response after transplantation operations and sirolimus- a bacterium-derived macrolide- used in the treatment of some cancers. The aim of this review is to analyze the current uses and the future applications in therapeutic treatments of microorganism-derived products (MdPs) and discuss the results obtained in the some clinical trials.

  7. The recombination epoch revisited

    NASA Technical Reports Server (NTRS)

    Krolik, Julian H.

    1989-01-01

    Previous studies of cosmological recombination have shown that this process produces as a by-product a highly superthermal population of Ly-alpha photons which retard completion of recombination. Cosmological redshifting was thought to determine the frequency distribution of the photons, while two-photon decay of hydrogen's 2s state was thought to control their numbers. It is shown here that frequency diffusion due to photon scattering dominate the cosmological redshift in the frequency range near line center which fixes the ratio of ground state to excited state population, while incoherent scattering into the far-red damping wing effectively destroys Ly-alpha photons as a rate which is competitive with two-photon decay. The former effect tends to hold back recombination, while the latter tends to accelerate it; the net results depends on cosmological parameters, particularly the combination Omega(b) h/sq rt (2q0), where Omega(b) is the fraction of the critical density provided by baryons.

  8. [Immobilized microorganisms and water purification].

    PubMed

    Mogilevich, N F

    1995-01-01

    Advantages and disadvantages of cells of aerobic microorganisms immobilized by the type of adhesion and incorporation into the gel beads, the amount of retained biomass, limitations of diffusion of oxygen and nutrients, viability, morphology, biochemical properties are described. Immobilized biocatalysts are discussed in the aspect of their use in purification of sewage waters.

  9. Smaller Fleas: Viruses of Microorganisms

    PubMed Central

    Hyman, Paul; Abedon, Stephen T.

    2012-01-01

    Life forms can be roughly differentiated into those that are microscopic versus those that are not as well as those that are multicellular and those that, instead, are unicellular. Cellular organisms seem generally able to host viruses, and this propensity carries over to those that are both microscopic and less than truly multicellular. These viruses of microorganisms, or VoMs, in fact exist as the world's most abundant somewhat autonomous genetic entities and include the viruses of domain Bacteria (bacteriophages), the viruses of domain Archaea (archaeal viruses), the viruses of protists, the viruses of microscopic fungi such as yeasts (mycoviruses), and even the viruses of other viruses (satellite viruses). In this paper we provide an introduction to the concept of viruses of microorganisms, a.k.a., viruses of microbes. We provide broad discussion particularly of VoM diversity. VoM diversity currently spans, in total, at least three-dozen virus families. This is roughly ten families per category—bacterial, archaeal, fungal, and protist—with some virus families infecting more than one of these microorganism major taxa. Such estimations, however, will vary with further discovery and taxon assignment and also are dependent upon what forms of life one includes among microorganisms. PMID:24278736

  10. Automated microorganism Sample Collection Module

    NASA Technical Reports Server (NTRS)

    Gall, L. S.; Graham, M. D.; Umbreit, W.

    1969-01-01

    Modified Gelman Sampler obtains representative sample of microorganism population. Proposed Sample Collection Module is based on direct inoculation of selected solid growth media encased in a cartridge at all times except during inoculation. Cartridge can be handled with no danger of contamination to sample or operator.

  11. L-methionine degradation potentialities of cheese-ripening microorganisms.

    PubMed

    Bonnarme, P; Lapadatescu, C; Yvon, M; Spinnler, H E

    2001-11-01

    Volatile sulphur compounds are major flavouring compounds in many traditional fermented foods including cheeses. These compounds are products of the catabolism of L-methionine by cheese-ripening microorganisms. The diversity of L-methionine degradation by such microorganisms, however, remains to be characterized. The objective of this work was to compare the capacities to produce volatile sulphur compounds by five yeasts, Geotrichum candidum, Yarrowia lipolytica, Kluyveromyces lactis, Debaryomyces hansenii, Saccharomyces cerevisiae and five bacteria, Brevibacterium linens, Corynebacterium glutamicum, Arthrobacter sp., Micrococcus lutens and Staphylococcus equorum of technological interest for cheese-ripening. The ability of whole cells of these microorganisms to generate volatile sulphur compounds from L-methionine was compared. The microorganisms produced a wide spectrum of sulphur compounds including methanethiol, dimethylsulfide, dimethyldisulfide, dimethyltrisulfide and also S-methylthioesters, which varied in amount and type according to strain. Most of the yeasts produced methanethiol, dimethylsulfide, dimethyldisulfide and dimethyltrisulfide but did not produce S-methylthioesters, apart from G. candidum that produced S-methyl thioacetate. Bacteria, especially Arth. sp. and Brevi. linens, produced the highest amounts and the greatest variety of volatile sulphur compounds includling methanethiol, sulfides and S-methylthioesters, e.g. S-methyl thioacetate, S-methyl thiobutyrate, S-methyl thiopropionate and S-methyl thioisovalerate. Cell-free extracts of all the yeasts and bacteria were examined for the activity of enzymes possibly involved in L-methionine catabolism, i.e. L-methionine demethiolase, L-methionine aminotransferase and L-methionine deaminase. They all possessed L-methionine demethiolase activity, while some (K. lactis, Deb. hansenii, Arth. sp., Staph. equorum) were deficient in L-methionine aminotransferase, and none produced L-methionine deaminase

  12. Ao38, a new cell line from eggs of the black witch moth, Ascalapha odorata (Lepidoptera: Noctuidae), is permissive for AcMNPV infection and produces high levels of recombinant proteins

    PubMed Central

    2010-01-01

    Background The insect cell line is a critical component in the production of recombinant proteins in the baculovirus expression system and new cell lines hold the promise of increasing both quantity and quality of protein production. Results Seventy cell lines were established by single-cell cloning from a primary culture of cells derived from eggs of the black witch moth (Ascalapha odorata; Lepidoptera, Noctuidae). Among 8 rapidly growing lines, cell line 38 (Ao38) was selected for further analysis, based on susceptibility to AcMNPV infection and production of secreted alkaline phosphatase (SEAP) from a baculovirus expression vector. In comparisons with low-passage High Five (BTI-Tn-5B1-4) cells, infected Ao38 cells produced β-galactosidase and SEAP at levels higher (153% and 150%, respectively) than those measured from High Five cells. Analysis of N-glycans of SEAP produced in Ao38 cells revealed two N-glycosylation sites and glycosylation patterns similar to those reported for High Five and Sf9 cells. Glycopeptide isoforms consisted of pauci- or oligomannose, with and without fucose on N-acetylglucosamine(s) linked to asparagine residues. Estimates of Ao38 cell volume suggest that Ao38 cells are approximately 2.5× larger than Sf9 cells but only approximately 74% of the size of High Five cells. Ao38 cells were highly susceptible to AcMNPV infection, similar to infectivity of Sf9 cells. Production of infectious AcMNPV budded virions from Ao38 cells peaked at approximately 4.5 × 107 IU/ml, exceeding that from High Five cells while lower than that from Sf9 cells. Ao38 cells grew rapidly in stationary culture with a population doubling time of 20.2 hr, and Ao38 cells were readily adapted to serum-free medium (Sf-900III) and to a suspension culture system. Analysis of Ao38 and a parental Ascalapha odorata cell line indicated that these lines were free of the alphanodavirus that was recently identified as an adventitious agent in High Five cell lines. Conclusions Ao

  13. Recombinant Vaccinia Virus: Immunization against Multiple Pathogens

    NASA Astrophysics Data System (ADS)

    Perkus, Marion E.; Piccini, Antonia; Lipinskas, Bernard R.; Paoletti, Enzo

    1985-09-01

    The coding sequences for the hepatitis B virus surface antigen, the herpes simplex virus glycoprotein D, and the influenza virus hemagglutinin were inserted into a single vaccinia virus genome. Rabbits inoculated intravenously or intradermally with this polyvalent vaccinia virus recombinant produced antibodies reactive to all three authentic foreign antigens. In addition, the feasibility of multiple rounds of vaccination with recombinant vaccinia virus was demonstrated.

  14. Recombinant Collagenlike Proteins

    NASA Technical Reports Server (NTRS)

    Fertala, Andzej

    2007-01-01

    A group of collagenlike recombinant proteins containing high densities of biologically active sites has been invented. The method used to express these proteins is similar to a method of expressing recombinant procollagens and collagens described in U. S. Patent 5,593,859, "Synthesis of human procollagens and collagens in recombinant DNA systems." Customized collagenous proteins are needed for biomedical applications. In particular, fibrillar collagens are attractive for production of matrices needed for tissue engineering and drug delivery. Prior to this invention, there was no way of producing customized collagenous proteins for these and other applications. Heretofore, collagenous proteins have been produced by use of such biological systems as yeasts, bacteria, and transgenic animals and plants. These products are normal collagens that can also be extracted from such sources as tendons, bones, and hides. These products cannot be made to consist only of biologically active, specific amino acid sequences that may be needed for specific applications. Prior to this invention, it had been established that fibrillar collagens consist of domains that are responsible for such processes as interaction with cells, binding of growth factors, and interaction with a number of structural proteins present in the extracellular matrix. A normal collagen consists of a sequence of domains that can be represented by a corresponding sequence of labels, e.g., D1D2D3D4. A collagenlike protein of the present invention contains regions of collagen II that contain multiples of a single domain (e.g., D1D1D1D1 or D4D4D4D4) chosen for its specific biological activity. By virtue of the multiplicity of the chosen domain, the density of sites having that specific biological activity is greater than it is in a normal collagen. A collagenlike protein according to this invention can thus be made to have properties that are necessary for tissue engineering.

  15. Recombinant allergens for specific immunotherapy.

    PubMed

    Cromwell, Oliver; Häfner, Dietrich; Nandy, Andreas

    2011-04-01

    Recombinant DNA technology provides the means for producing allergens that are equivalent to their natural counterparts and also genetically engineered variants with reduced IgE-binding activity. The proteins are produced as chemically defined molecules with consistent structural and immunologic properties. Several hundred allergens have been cloned and expressed as recombinant proteins, and these provide the means for making a very detailed diagnosis of a patient's sensitization profile. Clinical development programs are now in progress to assess the suitability of recombinant allergens for both subcutaneous and sublingual immunotherapy. Recombinant hypoallergenic variants, which are developed with the aim of increasing the doses that can be administered while at the same time reducing the risks for therapy-associated side effects, are also in clinical trials for subcutaneous immunotherapy. Grass and birch pollen preparations have been shown to be clinically effective, and studies with various other allergens are in progress. Personalized or patient-tailored immunotherapy is still a very distant prospect, but the first recombinant products based on single allergens or defined mixtures could reach the market within the next 5 years. Copyright © 2011 American Academy of Allergy, Asthma & Immunology. Published by Mosby, Inc. All rights reserved.

  16. Human Insulin from Recombinant DNA Technology

    NASA Astrophysics Data System (ADS)

    Johnson, Irving S.

    1983-02-01

    Human insulin produced by recombinant DNA technology is the first commercial health care product derived from this technology. Work on this product was initiated before there were federal guidelines for large-scale recombinant DNA work or commercial development of recombinant DNA products. The steps taken to facilitate acceptance of large-scale work and proof of the identity and safety of such a product are described. While basic studies in recombinant DNA technology will continue to have a profound impact on research in the life sciences, commercial applications may well be controlled by economic conditions and the availability of investment capital.

  17. Expression of recombinant antibodies.

    PubMed

    Frenzel, André; Hust, Michael; Schirrmann, Thomas

    2013-01-01

    Recombinant antibodies are highly specific detection probes in research, diagnostics, and have emerged over the last two decades as the fastest growing class of therapeutic proteins. Antibody generation has been dramatically accelerated by in vitro selection systems, particularly phage display. An increasing variety of recombinant production systems have been developed, ranging from Gram-negative and positive bacteria, yeasts and filamentous fungi, insect cell lines, mammalian cells to transgenic plants and animals. Currently, almost all therapeutic antibodies are still produced in mammalian cell lines in order to reduce the risk of immunogenicity due to altered, non-human glycosylation patterns. However, recent developments of glycosylation-engineered yeast, insect cell lines, and transgenic plants are promising to obtain antibodies with "human-like" post-translational modifications. Furthermore, smaller antibody fragments including bispecific antibodies without any glycosylation are successfully produced in bacteria and have advanced to clinical testing. The first therapeutic antibody products from a non-mammalian source can be expected in coming next years. In this review, we focus on current antibody production systems including their usability for different applications.

  18. Expression of Recombinant Antibodies

    PubMed Central

    Frenzel, André; Hust, Michael; Schirrmann, Thomas

    2013-01-01

    Recombinant antibodies are highly specific detection probes in research, diagnostics, and have emerged over the last two decades as the fastest growing class of therapeutic proteins. Antibody generation has been dramatically accelerated by in vitro selection systems, particularly phage display. An increasing variety of recombinant production systems have been developed, ranging from Gram-negative and positive bacteria, yeasts and filamentous fungi, insect cell lines, mammalian cells to transgenic plants and animals. Currently, almost all therapeutic antibodies are still produced in mammalian cell lines in order to reduce the risk of immunogenicity due to altered, non-human glycosylation patterns. However, recent developments of glycosylation-engineered yeast, insect cell lines, and transgenic plants are promising to obtain antibodies with “human-like” post-translational modifications. Furthermore, smaller antibody fragments including bispecific antibodies without any glycosylation are successfully produced in bacteria and have advanced to clinical testing. The first therapeutic antibody products from a non-mammalian source can be expected in coming next years. In this review, we focus on current antibody production systems including their usability for different applications. PMID:23908655

  19. Microorganisms in inorganic chemical analysis.

    PubMed

    Godlewska-Zyłkiewicz, Beata

    2006-01-01

    There are innumerable strains of microbes (bacteria, yeast and fungi) that degrade or transform chemicals and compounds into simpler, safer or less toxic substances. These bioprocesses have been used for centuries in the treatment of municipal wastes, in wine, cheese and bread making, and in bioleaching and metal recovery processes. Recent literature shows that microorganisms can be also used as effective sorbents for solid phase extraction procedures. This review reveals that fundamental nonanalytical studies on the parameters and conditions of biosorption processes and on metal-biomass interactions often result in efficient analytical procedures and biotechnological applications. Some selected examples illustrate the latest developments in the biosorption of metals by microbial biomass, which have opened the door to the application of microorganisms to analyte preconcentration, matrix separation and speciation analysis.

  20. Drug resistance in eukaryotic microorganisms.

    PubMed

    Fairlamb, Alan H; Gow, Neil A R; Matthews, Keith R; Waters, Andrew P

    2016-06-24

    Eukaryotic microbial pathogens are major contributors to illness and death globally. Although much of their impact can be controlled by drug therapy as with prokaryotic microorganisms, the emergence of drug resistance has threatened these treatment efforts. Here, we discuss the challenges posed by eukaryotic microbial pathogens and how these are similar to, or differ from, the challenges of prokaryotic antibiotic resistance. The therapies used for several major eukaryotic microorganisms are then detailed, and the mechanisms that they have evolved to overcome these therapies are described. The rapid emergence of resistance and the restricted pipeline of new drug therapies pose considerable risks to global health and are particularly acute in the developing world. Nonetheless, we detail how the integration of new technology, biological understanding, epidemiology and evolutionary analysis can help sustain existing therapies, anticipate the emergence of resistance or optimize the deployment of new therapies.

  1. Phosphate Biomineralization of Cambrian Microorganisms

    NASA Technical Reports Server (NTRS)

    McKay, David S.; Rozanov, Alexei Yu.; Hoover, Richard B.; Westall, Frances

    1998-01-01

    As part of a long term study of biological markers (biomarkers), we are documenting a variety of features which reflect the previous presence of living organisms. As we study meteorites and samples returned from Mars, our main clue to recognizing possible microbial material may be the presence of biomarkers rather than the organisms themselves. One class of biomarkers consists of biominerals which have either been precipitated directly by microorganisms, or whose precipitation has been influenced by the organisms. Such microbe-mediated mineral formation may include important clues to the size, shape, and environment of the microorganisms. The process of fossilization or mineralization can cause major changes in morphologies and textures of the original organisms. The study of fossilized terrestrial organisms can help provide insight into the interpretation of mineral biomarkers. This paper describes the results of investigations of microfossils in Cambrian phosphate-rich rocks (phosphorites) that were found in Khubsugul, Northern Mongolia.

  2. Drug resistance in eukaryotic microorganisms

    PubMed Central

    Fairlamb, Alan H.; Gow, Neil A. R.; Matthews, Keith R.; Waters, Andrew P.

    2016-01-01

    Eukaryotic microbial pathogens are major contributors to illness and death globally. Although much of their impact can be controlled by drug therapy as with prokaryotic microorganisms, the emergence of drug resistance has threatened these treatment efforts. Here, we discuss the challenges posed by eukaryotic microbial pathogens and how these are similar to, or differ from, the challenges of prokaryotic antibiotic resistance. The therapies used for several major eukaryotic microorganisms are then detailed, and the mechanisms that they have evolved to overcome these therapies are described. The rapid emergence of resistance and the restricted pipeline of new drug therapies pose considerable risks to global health and are particularly acute in the developing world. Nonetheless, we detail how the integration of new technology, biological understanding, epidemiology and evolutionary analysis can help sustain existing therapies, anticipate the emergence of resistance or optimize the deployment of new therapies. PMID:27572976

  3. Microorganisms resistant to free-living amoebae.

    PubMed

    Greub, Gilbert; Raoult, Didier

    2004-04-01

    Free-living amoebae feed on bacteria, fungi, and algae. However, some microorganisms have evolved to become resistant to these protists. These amoeba-resistant microorganisms include established pathogens, such as Cryptococcus neoformans, Legionella spp., Chlamydophila pneumoniae, Mycobacterium avium, Listeria monocytogenes, Pseudomonas aeruginosa, and Francisella tularensis, and emerging pathogens, such as Bosea spp., Simkania negevensis, Parachlamydia acanthamoebae, and Legionella-like amoebal pathogens. Some of these amoeba-resistant bacteria (ARB) are lytic for their amoebal host, while others are considered endosymbionts, since a stable host-parasite ratio is maintained. Free-living amoebae represent an important reservoir of ARB and may, while encysted, protect the internalized bacteria from chlorine and other biocides. Free-living amoebae may act as a Trojan horse, bringing hidden ARB within the human "Troy," and may produce vesicles filled with ARB, increasing their transmission potential. Free-living amoebae may also play a role in the selection of virulence traits and in adaptation to survival in macrophages. Thus, intra-amoebal growth was found to enhance virulence, and similar mechanisms seem to be implicated in the survival of ARB in response to both amoebae and macrophages. Moreover, free-living amoebae represent a useful tool for the culture of some intracellular bacteria and new bacterial species that might be potential emerging pathogens.

  4. Microorganisms Resistant to Free-Living Amoebae

    PubMed Central

    Greub, Gilbert; Raoult, Didier

    2004-01-01

    Free-living amoebae feed on bacteria, fungi, and algae. However, some microorganisms have evolved to become resistant to these protists. These amoeba-resistant microorganisms include established pathogens, such as Cryptococcus neoformans, Legionella spp., Chlamydophila pneumoniae, Mycobacterium avium, Listeria monocytogenes, Pseudomonas aeruginosa, and Francisella tularensis, and emerging pathogens, such as Bosea spp., Simkania negevensis, Parachlamydia acanthamoebae, and Legionella-like amoebal pathogens. Some of these amoeba-resistant bacteria (ARB) are lytic for their amoebal host, while others are considered endosymbionts, since a stable host-parasite ratio is maintained. Free-living amoebae represent an important reservoir of ARB and may, while encysted, protect the internalized bacteria from chlorine and other biocides. Free-living amoebae may act as a Trojan horse, bringing hidden ARB within the human “Troy,” and may produce vesicles filled with ARB, increasing their transmission potential. Free-living amoebae may also play a role in the selection of virulence traits and in adaptation to survival in macrophages. Thus, intra-amoebal growth was found to enhance virulence, and similar mechanisms seem to be implicated in the survival of ARB in response to both amoebae and macrophages. Moreover, free-living amoebae represent a useful tool for the culture of some intracellular bacteria and new bacterial species that might be potential emerging pathogens. PMID:15084508

  5. DIALYSIS FLASK FOR CONCENTRATED CULTURE OF MICROORGANISMS

    PubMed Central

    Gerhardt, Philipp; Gallup, D. M.

    1963-01-01

    Gerhardt, Philipp (The University of Michigan, Ann Arbor), and D. M. Gallup. Dialysis flask for concentrated culture of microorganisms. J. Bacteriol 86:919–929. 1963.—A twin-chambered dialysis flask was designed with a supported membrane clamped between a reservoir of medium in the bottom and a small volume of culture above, the unit being mounted on a shaking machine to provide aeration and agitation. The performance of different dialysis membranes and membrane filters was compared in glucose-diffusion and bacterial-culture tests. Some of the variables in dialysis culture were assessed and the growth response was characterized, with Serratia marcescens as the test organism. The general usefulness and concentrating effect of dialysis culture were demonstrated in trials with 16 representative types of microorganisms. Dialysis culture was shown to be especially suitable for producing dense populations of cells or their macromolecular products in an environment free from complex medium constituents, for removing toxic products that limit growth or fermentation, and for supplying oxygen by diffusion without the damage from usual aeration procedures. Images PMID:14080802

  6. Biomachining: metal etching via microorganisms.

    PubMed

    Díaz-Tena, Estíbaliz; Barona, Astrid; Gallastegui, Gorka; Rodríguez, Adrián; López de Lacalle, L Norberto; Elías, Ana

    2017-05-01

    The use of microorganisms to remove metal from a workpiece is known as biological machining or biomachining, and it has gained in both importance and scientific relevance over the past decade. Conversely to mechanical methods, the use of readily available microorganisms is low-energy consuming, and no thermal damage is caused during biomachining. The performance of this sustainable process is assessed by the material removal rate, and certain parameters have to be controlled for manufacturing the machined part with the desired surface finish. Although the variety of microorganisms is scarce, cell concentration or density plays an important role in the process. There is a need to control the temperature to maintain microorganism activity at its optimum, and a suitable shaking rate provides an efficient contact between the workpiece and the biological medium. The system's tolerance to the sharp changes in pH is quite limited, and in many cases, an acid medium has to be maintained for effective performance. This process is highly dependent on the type of metal being removed. Consequently, the operating parameters need to be determined on a case-by-case basis. The biomachining time is another variable with a direct impact on the removal rate. This biological technique can be used for machining simple and complex shapes, such as series of linear, circular, and square micropatterns on different metal surfaces. The optimal biomachining process should be fast enough to ensure high production, a smooth and homogenous surface finish and, in sum, a high-quality piece. As a result of the high global demand for micro-components, biomachining provides an effective and sustainable alternative. However, its industrial-scale implementation is still pending.

  7. Minerals and Microorganisms in Evaporite Environments

    NASA Astrophysics Data System (ADS)

    Morris, P. A.; Brigmon, R. L.

    2010-12-01

    Traditional analysis of evaporite environments have either focused on the geology or the halophilic organisms. It is relatively rare that the two have been combined and even rarer that both disciplines have been incorporated in comparing evaporite sites. The variation in evaporite environments does influence microbial ecology and fossilization processes as each site varies in pH, temperature, presence or absence springs, and spring chemistry. Understanding the evaporite environments is important for planetary scientists as they serve as analogs for evaluating extraterrestrial materials, including the potential for water and ultimately life. For example Mars lander, rover and orbital missions have identified the evaporite signatures of gypsum, carbonates and chlorides, all indicating that water existed at sometime in the planets geological history. Terrestrial evaporite sites all possess halophilic tolerant life. In some instances such as the Dead Sea, Israel, it is restricted to microbial life, but in other sites there are higher life forms. The microbes associated with these evaporite sites can produce biofilms as a method to develop their own microenvironments. Microorganisms can be observed colonizing specific ecological niches or gradients can be created by these environments. These gradients occur due the localized drying and weathering patterns that create different soil chemistry. The microorganisms in turn colonize specific areas more suitable to their specific metabolic needs. For example, under anaerobic conditions with sulfur and methane prevalent methanogenic and/or sulfur reducing microbial species may be observed. However, under similar chemistry environments with the exception of aerobic conditions sulfur oxidizer and/or methanotrophic microorganism may occur. Because of their conspicuous colored pigments purple sulfur bacteria are frequently observed in anoxic zones of lakes, sulfur springs, and stratified evaporite crusts. Some of these bacteria

  8. Studying marine microorganisms from space.

    PubMed

    Pedrós-Alió, C; Simó, R

    2002-12-01

    Microorganisms are but a few micrometers in diameter and are not visible to the naked eye. Yet, the large numbers of microorganisms present in the oceans and the global impact of their activities make it possible to observe them from space. Here a few examples of how microorganisms can be studied from satellites are presented. The first case is the best known: the main pigment used in photosynthesis (chlorophyll a) can be determined from satellites. These kinds of studies have contributed a tremendous amount of understanding about the distribution and dynamics of primary production in the oceans. Two other examples will concern analysis of heterotrophic prokaryotic production and estimates of dimethyl sulfide (DMS) concentration and flux to the atmosphere. These three processes are of fundamental importance for the functioning of the biosphere. Marine microbes carry out about half of the total primary production in the planet. A substantial fraction of the respiration in the oceans is due to the activity of heterotrophic prokaryotes. Finally, the flux of DMS to the atmosphere is believed to constitute one of the mechanisms by which the biota can regulate climate. The global implications of microbial processes in the oceans can only be addressed with the help of satellites.

  9. Microorganisms in closed periapical lesions.

    PubMed

    Abou-Rass, M; Bogen, G

    1998-01-01

    The purpose of this study was to investigate the microorganisms of strictly selected closed periapical lesions associated with both refractory endodontic therapy and pulpal calcification. Definitive criteria were established that assured complete clinical isolation of the periapical lesion from the oral and periodontal environment. A total of 13 criteria-referenced lesions were selected from 70 patients with endodontic surgical indications. A well controlled culturing method was used in all cases and samples were taken by one clinician at three separate sites during each surgery. Samples taken at the surgical window and within the body of the lesion served as controls, whilst a third sample was taken at the apex. In all 13 cases, samples taken from the apex yielded microorganisms comprising 63.6% obligate anaerobes and 36.4% facultative anaerobes. Prevalence of the isolated species was 31.8% for Actinomyces sp., 22.7% Propionibacterium sp., 18.2% Streptococcus sp., 13.6% Staphlyococcus sp., 4.6% Porphyromonas gingivalis, 4.6% Peptostreptococcus micros and 4.6% Gram-negative enterics. The results of this investigation indicate that closed periapical lesions associated with calcified teeth or those resistant to root canal treatment harbour bacteria. The inability to eradicate all root canal microorganisms during root canal treatment, along with anatomical factors, may allow further bacterial colonization of the root apex and surrounding periapical tissues, and consequently prevent healing.

  10. [Mycelial fungi maintained in the Russia Collection of Microorganisms (VKM IBPM RAS)].

    PubMed

    Ozerskaia, S M; Kochkina, G A; Ivanushkina, N E; Zaprometova, K M; Eremina, S S

    2005-01-01

    Information on application of diversity of mycelial fungi maintained in the Russia Collection of Microorganisms (VKM) at the Institute for Biochemistry and Physiology of Microorganisms, Russian Academy of Sciences to research and biotechnology as producers is presented, as well as data on additions to the basic collection.

  11. [Questions safety and tendency of using genetically modified microorganisms in food, food additives and food derived].

    PubMed

    Khovaev, A A

    2008-01-01

    In this article analysis questions of using genetically modified microorganisms in manufacture food production, present new GMM used in manufacture -food ferments; results of medical biological appraisal/ microbiological and genetic expert examination/ of food, getting by use microorganisms or there producents with indication modern of control methods.

  12. Predatory Microorganisms Would Help Reclaim Water

    NASA Technical Reports Server (NTRS)

    Benjaminson, Morris A.; Lehrer, Stanley

    1995-01-01

    Wastewater-reclamation systems of proposed type use predatory, nonpathogenic microorganisms to consume pathogenic microorganisms. Unlike some other wastewater-reclamation systems, these systems do not require use of toxic chemicals, intense heat, or ionizing radiation (conductivity rays or ultraviolet) to destroy microorganisms.

  13. 40 CFR 725.85 - Microorganism identity.

    Code of Federal Regulations, 2013 CFR

    2013-07-01

    ... 40 Protection of Environment 32 2013-07-01 2013-07-01 false Microorganism identity. 725.85 Section... ACT REPORTING REQUIREMENTS AND REVIEW PROCESSES FOR MICROORGANISMS Confidentiality and Public Access to Information § 725.85 Microorganism identity. (a) Claims applicable to the period prior to...

  14. 40 CFR 725.85 - Microorganism identity.

    Code of Federal Regulations, 2014 CFR

    2014-07-01

    ... 40 Protection of Environment 31 2014-07-01 2014-07-01 false Microorganism identity. 725.85 Section... ACT REPORTING REQUIREMENTS AND REVIEW PROCESSES FOR MICROORGANISMS Confidentiality and Public Access to Information § 725.85 Microorganism identity. (a) Claims applicable to the period prior to...

  15. 40 CFR 725.85 - Microorganism identity.

    Code of Federal Regulations, 2010 CFR

    2010-07-01

    ... 40 Protection of Environment 30 2010-07-01 2010-07-01 false Microorganism identity. 725.85 Section... ACT REPORTING REQUIREMENTS AND REVIEW PROCESSES FOR MICROORGANISMS Confidentiality and Public Access to Information § 725.85 Microorganism identity. (a) Claims applicable to the period prior to...

  16. 40 CFR 725.85 - Microorganism identity.

    Code of Federal Regulations, 2012 CFR

    2012-07-01

    ... 40 Protection of Environment 32 2012-07-01 2012-07-01 false Microorganism identity. 725.85 Section... ACT REPORTING REQUIREMENTS AND REVIEW PROCESSES FOR MICROORGANISMS Confidentiality and Public Access to Information § 725.85 Microorganism identity. (a) Claims applicable to the period prior to...

  17. 40 CFR 725.85 - Microorganism identity.

    Code of Federal Regulations, 2011 CFR

    2011-07-01

    ... 40 Protection of Environment 31 2011-07-01 2011-07-01 false Microorganism identity. 725.85 Section... ACT REPORTING REQUIREMENTS AND REVIEW PROCESSES FOR MICROORGANISMS Confidentiality and Public Access to Information § 725.85 Microorganism identity. (a) Claims applicable to the period prior to...

  18. Predatory Microorganisms Would Help Reclaim Water

    NASA Technical Reports Server (NTRS)

    Benjaminson, Morris A.; Lehrer, Stanley

    1995-01-01

    Wastewater-reclamation systems of proposed type use predatory, nonpathogenic microorganisms to consume pathogenic microorganisms. Unlike some other wastewater-reclamation systems, these systems do not require use of toxic chemicals, intense heat, or ionizing r