1,721,153 research outputs found
Activation of formate hydrogen-lyase via expression of uptake [NiFe]-hydrogenase in Escherichia coli BL21(DE3)
Full text linkBackground: Several recent studies have reported successful hydrogen (H-2) production achieved via recombinant expression of uptake [NiFe]-hydrogenases from Hydrogenovibrio marinus, Rhodobacter sphaeroides, and Escherichia coli (hydrogenase-1) in E. coli BL21(DE3), a strain that lacks H-2-evolving activity. However, there are some unclear points that do not support the conclusion that the recombinant hydrogenases are responsible for the in vivo H-2 production. Results: Unlike wild-type BL21(DE3), the recombinant BL21(DE3) strains possessed formate hydrogen-lyase (FHL) activities. Through experiments using fdhF (formate dehydrogenase-H) or hycE (hydrogenase-3) mutants, it was shown that H-2 production was almost exclusively dependent on FHL. Upon expression of hydrogenase, extracellular formate concentration was changed even in the mutant strains lacking FHL, indicating that formate metabolism other than FHL was also affected. The two subunits of H. marinus uptake [NiFe]-hydrogenase could activate FHL independently of each other, implying the presence of more than two different mechanisms for FHL activation in BL21(DE3). It was also revealed that the signal peptide in the small subunit was essential for activation of FHL via the small subunit. Conclusions: Herein, we demonstrated that the production of H-2 was indeed induced via native FHL activated by the expression of recombinant hydrogenases. The recombinant strains with [NiFe]-hydrogenase appear to be unsuitable for practical in vivo H-2 production due to their relatively low H-2 yields and productivities. We suggest that an improved H-2-producing cell factory could be designed by constructing a well characterized and overproduced synthetic H-2 pathway and fully activating the native FHL in BL21(DE3).1122Ysciescopu
Facile Evaluation of Cell Disruption Efficiency Using pH-Controlled Fluorescence Resonance Energy Transfer
No full textThe evaluation of cell disruption efficiency is important in monitoring durability and diverse cellular physiologies of host cells and improving recovery yield of intracellular products. On the basis of our previously suggested pH-controlled fluorescence resonance energy transfer (FRET) system, we herein demonstrated that easy and effective estimation of the degree of cell disruption under certain stress environments is possible using a simple measurement of changes in fluorescence spectra of two fused fluorescent proteins, because its unique linear correlation was established with cell disruption efficiency from the conventional colony counting method.X112sciescopu
Observation and modeling of induction effect on human transferrin production from stably transfected Drosophila S2 cell culture
No full textHuman transferrin (hTf) is a serum glycoprotein involved in iron transport. We performed, for the first time, mathematical modeling of stably transfected insect Drosophila melanogaster S2 cell culture, a nonlytic plasmid-based system that secretes recombinant hTf under control of the copper sulfate-inducible Drosophila metallothionein promoter. Cell growth patterns at various inducer concentrations revealed that the specific growth rate of S2 cells was substantially reduced as the specific rate of recombinant hTf production increased, and recovered to some extent when recombinant hTf production was nearly stopped. Additionally, the time profiles of specific production rates exhibited a maximum in the early culture period. Longer times and lower values of the maximum specific production rate were observed at lower inducer concentrations, and shorter times and higher values at higher inducer levels. Although the proposed S2 cell culture model was slightly limited with regard to prediction of cell growth profile at the late stage, we ensured that it gave reasonable predictions of the dynamics of glucose consumption and recombinant hTf production and confirmed its validity through simulating other culture experiments under different conditions. The S2 model proposed in this study can contribute to the elucidation of cell culture dynamics, and optimization of each culture variable to enhance heterologous protein production. (c) 2005 Elsevier Inc. All rights reserved.X1112sciescopu
Pattern-Mapped Multiple Detection of 11 Pathogenic Bacteria Using a 16S rDNA-Based Oligonucleotide Microarray
No full textPathogen detection is an important issue in human health due to the threats posed by severe communicable diseases. In the present study, to achieve efficient and accurate multiple detection of 11 selected pathogenic bacteria, we constructed a 16S rDNA oligonucleotide microarray containing doubly specific capture probes. Many target pathogens were specifically detected by the microarray with the aid of traditional perfect match-based analysis using our previously proposed two-dimensional visualization plot tool. However, some target species or subtypes were difficult to discriminate by perfect match analysis due to nonspecific binding of conserved 16S rDNA-derived capture probes with high sequence similarity. We noticed that the patterns of specific spots for each strain were somewhat different in the two-dimensional gradation plot. Therefore, to discriminate subtle differences between phylogenetically related pathogens, a pattern-mapping statistical model was established using an artificial neural network algorithm trained by experimental repeats. The oligonucleotide microarray system harboring doubly specific capture probes combined with the pattern-mapping analysis tool resulted in successful detection of all target pathogens including even subtypes of two closely related species showing strong nonspecific binding. Collectively, the results indicate that our novel combined system of a 16S rDNA-based DNA microarray and a pattern-mapping statistical analysis tool is a simple and effective method for detecting multiple pathogens. Biotechnol. Bioeng. 2010;106: 183-192. (C) 2010 Wiley Periodicals, Inc.X111010sciescopu
Quantitative oligonucleotide microarray data analysis with an artificial standard probe strategy
No full textQuantitative data analysis is an important element in several applications of DNA microarray, including mRNA expression profiling and estimation of infectious doses for pathogens. Here, we introduce an artificial standard probe strategy for quantitative pathogen detection using an oligonucleotide chip as a model system. The standard capture probe sequence was artificially designed to prevent non-specific hybridization with bacterial targets. Based on the fluorescence intensities of artificial standard spots, the raw fluorescence intensity data for specific spots could be corrected to generate linear correlations with target concentrations. Therefore, our novel artificial standard probe may be effectively applied for the correction of chip-to-chip variations and quantitative data analysis of a one-color labeled DNA microarray system. (c) 2008 Elsevier B.V. All rights reserved.X1111sciescopu
Recent progress in hydrogenase and its biotechnological application for viable hydrogen technology
No full textDespite increasing interest in hydrogen (H-2) as an alternative energy carrier, the current production of H-2 still depends on fossil fuels. Biotechnological hydrogen production can provide a more sustainable way to generate H-2. Hydrogenases are key enzymes involved in hydrogen metabolism of microorganisms with roles of H-2 oxidation or evolution. They have potential applications in H-2 production in vivo, in vitro and fuel cell. Important achievements have been made over the past decade in our understanding of hydrogenase and its biotechnological application as catalyst for H-2 production and fuel cell. This review summarizes recent progress in the study of hydrogenases, involving strategies for biosynthesis, maturation process, isolation of novel hydrogenases, heterologous expression system, structural feature of oxygen (O-2)-tolerant hydrogenases, and biotechnological applications for viable H-2 technology.X111110sciescopuskc
Statistical optimization for immobilized metal affinity purification of secreted human erythropoietin from Drosophila S2 cells
No full textWe used a novel approach to affinity purify human erythropoietin (hEPO) following its secretion from Drosophila melanogaster S2 cells. Immobilized metal affinity purification of hEPO was optimized using a two-step serial statistical optimization strategy. After determining the elution conditions (based on preliminary batch-type purification experiments), the first optimization step considered three purification factors; resin, equilibrium, and washing. The results of this analysis showed that the resin amount was the major factor influencing yield and purity in both model equations and the washing factor lowered the confidence limits of the acquired model equations. The washing conditions were then set based on the results of the first step optimization and the second step then optimized three factors; resin, equilibrium, and elution. The yield and purity of hEPO were then compared following purification using three different approaches; batch-type purification based upon the conditions determined by serial statistical optimization, batch-type purification performed in preliminary experiments, and FPLC column chromatography-type purification. We found that the serial statistical optimization approach provided the best combination of yield and purity. These findings indicate that serial statistical optimization strategies can be successfully employed for immobilized metal affinity protein purification using either batch-type or column approaches. (C) 2002 Elsevier Science (USA). All rights reserved.X1118sciescopu
Solubility dependency of co-expression effects of stress-induced protein Dps on foreign protein expression in Escherichia coli
No full textThe over-expression of foreign proteins imposes metabolic burden on host strains that may lead to reduced cell growth and even yield of target protein. We investigated dependency of co-expression effects of stress-induced non-specific DNA-binding protein, Dps, on cell growth and foreign protein expression according to solubility in Escherichia coli. Dps co-expression showed clear distinct effects according to solubility of target proteins. Under co-expression of recombinant Dps, cell growth for the strain expressing baculoviral polyhedrin (Polh)-green fluorescent protein (GFP) fusion protein or human interleukin-2 (hIL-2) that were expressed as insoluble inclusion body had tendency to decline slightly compared to each Dps non-expressing strain. However, cell growth for the strain expressing soluble GFP or Mussel adhesive protein type 5 (Mgfp-5) was somewhat increased. While Dps co-expression had somewhat negative effects on expression of soluble protein, it showed huge impacts on product yield of insoluble proteins (1.6-1.8-fold for Polh-GFP and 4-5-fold for hIL-2). Therefore, it was obvious that co-expression of Dps has different effects on foreign protein production according to solubility. Proteomic analyses revealed that Dps co-expression induced significantly different global patterns through interaction with target foreign protein according to target solubility. These global pattern alterations might be favorable for production of insoluble foreign proteins indirectly. (c) 2006 Elsevier Inc. All rights reserved.X114sciescopu
Down-regulation of acetate pathway through antisense strategy in Escherichia coli: Improved foreign protein production
No full textA problem with the use of Escherichia coli to produce foreign proteins is that although endogenously produced acetate is physiologically indispensable, it inhibits protein expression. Here we firstly employed an antisense RNA strategy as an elaborate metabolic engineering tool to partially block biosynthesis of two major acetate pathway enzymes, phosphotransacetylase (PTA) and acetate kinase (ACK). Three recombinant plasmids containing antisense genes targeting either or both of pta and ackA were constructed, and their effects on the acetate pathway and foreign protein productivity compared to control plasmid without any antisense genes were determined in E. coli BL21. Green fluorescent protein (GFP) was employed as a model foreign protein, and timing of antisense expression was controlled by using the intrinsic ackA promoter. We found that the antisense method partially reduced mRNA levels of target enzyme genes and, over time, lowered the concentration of acetate in culture media in all antisense-regulated strains. Notably, total production of GFP was enhanced 1.6- to 2.1-fold in antisense-regulated strains, even though the degree of acetate reduction was not significantly large. It was revealed that the acetate pathway has more critical roles in cellular physiology than expected in the previous reports. When the scale of culture was increased, enhancement of protein production became larger, demonstrating that this antisense strategy can be successfully applied to practical large-scale protein production processes. (C) 2003 Wiley Periodicals, Inc.X114545sciescopu
Disperse Distribution of Cationic Amino Acids on Hydrophilic Surface of Helical Wheel Enhances Antimicrobial Peptide Activity
No full textThe antimicrobial action of amphipathic antimicrobial peptides (AMPs) generally depends on perturbation of the bacterial membrane via electrostatic interactions promoting initial binding to the surface and hydrophobic interactions for pore formation into the membrane. Several studies have focused on the structure-activity relationship (SAR) of AMPs by modulation of structural parameters. However, modulation of one parameter commonly induces simultaneous changes in other parameters, making it difficult to investigate the specific influence of a single variable. In the present work, we investigated the distribution effect of cationic amino acids on the hydrophilic surface of the helical wheel using model AMPs composed of only lysine (K) and leucine (L) as representative cationic and hydrophobic residues, respectively, under conditions in which other parameters are fixed. Based on SAR analyses of alpha-helical KL model AMPs displaying different cationic distributions, we propose that the dispersity of cationic amino acids on the hydrophilic surface is a factor that contributes to the antimicrobial activity of AMP. Moreover, antimicrobial activity is enhanced by rearrangement of cationic amino acids to promote dispersed distribution. We confirmed the cationic distribution effect using natural AMP-derived alpha-helical CRAMP18 and its analogs. Our data show that accumulation of lysine shifts in the CRAMP18 analog leads to higher dispersion, and subsequently to improved antimicrobial activity. Therefore, we propose that the cationic distribution effect can be applied for the rational redesign of amino acid sequences to improve the antimicrobial activities of natural alpha-helical AMPs, in combination with regulation of other known structural parameters. Biotechnol. Bioeng. 2010;107: 216-223. (c) 2010 Wiley Periodicals, Inc.X111415sciescopu
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