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1.  A novel adenoviral vector carrying an all-in-one Tet-On system with an autoregulatory loop for tight, inducible transgene expression 
BMC Biotechnology  2015;15(1):4.
One of the most commonly used vectors for gene therapy is the adenoviral vector; its ability to tightly regulate transgene expression is critical for optimizing therapeutic outcomes. The tetracycline-regulated system (especially the Tet-On system) for gene expression is one of the most valuable tools for controlling gene expression. The major problem of an adenoviral vector carrying a Tet-On system is suboptimal regulation of transgene expression.
We constructed a single adenoviral vector carrying in its E1 region a novel “all-in-one” Tet-On system with an autoregulatory loop. This system had improved Dox-inducible gene expression in terms of low basal expression, high induced expression and high responsiveness to Dox. To our knowledge, this is the first reported adenovirus-based, all-in-one Tet-On system with an autoregulatory loop inserted into a single region of adenoviral genome. This system was further tested by inducible expression of soluble tumor necrosis factor-related apoptosis-inducing ligand (sTRAIL). The adenovirus that expressed soluble TRAIL under the control of this novel Tet-On system showed tumor-derived cells inhibitory activity in SW480 cells only under induced conditions.
Our novel, single adenoviral vector carrying in its E1 region an all-in-one Tet-On system with an autoregulatory loop displayed tight regulation of transgene expression in vitro. This system has great potential for a variety of applications, including gene therapy and the study of gene function.
PMCID: PMC4331377
Adenoviral vector; Tet-On; Transgene; Inducible vector; Gene therapy
2.  Heterologous expression and biochemical characterization of a highly active and stable chloroplastic CuZn-superoxide dismutase from Pisum sativum 
BMC Biotechnology  2015;15(1):3.
CuZn-Superoxide dismutase (SOD) is a unique enzyme, which can catalyzes the dismutation of inevitable metabolic product i.e.; superoxide anion into molecular oxygen and hydrogen peroxide. The enzyme has gained wide interest in pharmaceutical industries due to its highly acclaimed antioxidative properties. The recombinant expression of this protein in its enzymatically active and stable form is highly desired and hence optimization of culture conditions and characterization of the related biochemical properties are essential to explore the significance of the enzyme in physiological, therapeutic, structural and transgenic research.
High-level expression of the chloroplastic isoform of Pisum sativum CuZn-SOD was achieved at 18°C, upon isopropyl β-D-1-thiogalactopyranoside induction and the process was optimized for maximum recovery of the protein in its soluble (enzymatically active) form. Both crude and purified protein fractions display significant increase in activity following supplementation of defined concentration Cu (CuSO4) and Zn (ZnSO4). Yield of the purified recombinant protein was ~ 4 mg L−1 of culture volume and the bacterial biomass was ~ 4.5 g L−1. The recombinant pea chloroplastic SOD was found to possess nearly 6 fold higher superoxide dismutase activity and the peroxidase activity was also 5 fold higher as compared to commercially available CuZn-superoxide dismutase. The computational, spectroscopic and biochemical characterization reveals that the protein harbors all the characteristics features of this class of enzyme. The enzyme was found to be exceptionally stable as evident from pH and temperature incubation studies and maintenance of SOD activity upon prolonged storage.
Overexpression and purification strategy presented here describes an efficient protocol for the production of a highly active and stable CuZn-superoxide dismutase in its recombinant form in E. coli system. The strategy can be utilized for the large-scale preparation of active CuZn-superoxide dismutase and thus it has wide application in pharmaceutical industries and also for elucidating the potential of this protein endowed with exceptional stability and activity.
Electronic supplementary material
The online version of this article (doi:10.1186/s12896-015-0117-0) contains supplementary material, which is available to authorized users.
PMCID: PMC4333176
Heterologous expression; CD spectroscopy; Homology modelling; Ni-NTA purification; Peroxidase activity; Superoxide dismutase
3.  Synthesis of Fructooligosaccharides by IslA4, a truncated inulosucrase from Leuconostoc citreum 
BMC Biotechnology  2015;15(1):2.
IslA4 is a truncated single domain protein derived from the inulosucrase IslA, which is a multidomain fructosyltransferase produced by Leuconostoc citreum. IslA4 can synthesize high molecular weight inulin from sucrose, with a residual sucrose hydrolytic activity. IslA4 has been reported to retain the product specificity of the multidomain enzyme.
Screening experiments to evaluate the influence of the reactions conditions, especially the sucrose and enzyme concentrations, on IslA4 product specificity revealed that high sucrose concentrations shifted the specificity of the reaction towards fructooligosaccharides (FOS) synthesis, which almost eliminated inulin synthesis and led to a considerable reduction in sucrose hydrolysis. Reactions with low IslA4 activity and a high sucrose activity allowed for high levels of FOS synthesis, where 70% sucrose was used for transfer reactions, with 65% corresponding to transfructosylation for the synthesis of FOS.
Domain truncation together with the selection of the appropriate reaction conditions resulted in the synthesis of various FOS, which were produced as the main transferase products of inulosucrase (IslA4). These results therefore demonstrate that bacterial fructosyltransferase could be used for the synthesis of inulin-type FOS.
PMCID: PMC4331173
Fructosyltransferase; Fructooligosaccharides; Inulin; Inulosucrase; Leuconostoc citreum
4.  A novel psychrophilic alkaline phosphatase from the metagenome of tidal flat sediments 
BMC Biotechnology  2015;15(1):1.
Alkaline phosphatase (AP) catalyzes the hydrolytic cleavage of phosphate monoesters under alkaline conditions and plays important roles in microbial ecology and molecular biology applications. Here, we report on the first isolation and biochemical characterization of a thermolabile AP from a metagenome.
The gene encoding a novel AP was isolated from a metagenomic library constructed with ocean-tidal flat sediments from the west coast of Korea. The metagenome-derived AP (mAP) gene composed of 1,824 nucleotides encodes a polypeptide with a calculated molecular mass of 64 kDa. The deduced amino acid sequence of mAP showed a high degree of similarity to other members of the AP family. Phylogenetic analysis revealed that the mAP is shown to be a member of a recently identified family of PhoX that is distinct from the well-studied classical PhoA family. When the open reading frame encoding mAP was cloned and expressed in recombinant Escherichia coli, the mature mAP was secreted to the periplasm and lacks an 81-amino-acid N-terminal Tat signal peptide. Mature mAP was purified to homogeneity as a monomeric enzyme with a molecular mass of 56 kDa. The purified mAP displayed typical features of a psychrophilic enzyme: high catalytic activity at low temperature and a remarkable thermal instability. The optimal temperature for the enzymatic activity of mAP was 37°C and complete thermal inactivation of the enzyme was observed at 65°C within 15 min. mAP was activated by Ca2+ and exhibited maximal activity at pH 9.0. Except for phytic acid and glucose 1-phosphate, mAP showed phosphatase activity against various phosphorylated substrates indicating that it had low substrate specificity. In addition, the mAP was able to remove terminal phosphates from cohesive and blunt ends of linearized plasmid DNA, exhibiting comparable efficiency to commercially available APs that have been used in molecular biology.
The presented mAP enzyme is the first thermolabile AP found in cold-adapted marine metagenomes and may be useful for efficient dephosphorylation of linearized DNA.
PMCID: PMC4335783  PMID: 25636680
Alkaline phosphatase; Metagenome; PhoX; Escherichia coli
5.  PHDs inhibitor DMOG promotes the vascularization process in the AV loop by HIF-1a up-regulation and the preliminary discussion on its kinetics in rat 
BMC Biotechnology  2014;14(1):112.
The Arterovenous Loop (AV Loop) model is a vascularization model in tissue engineering research, which is capable of generating a three dimensional in vivo unit with cells as well as the supporting vessels within an isolation chmaber. In our previous studies the AV loop in the isolation chamber was discovered to undergo hypoxia, characterized by Hypoxia Inducible Factor (HIF) up-regulation. The vascularization followed the increase of HIF-α temporally, while it was spatially positively correlated with the HIF-α level, as well. This study aims to prove that HIF-1a up-regulation is the stimulus for vascularization in the AV loop model.
The AV loop model in rats was created by interposing a femoral vein graft into the distal ends of the contralateral femoral artery and vein, and the loop was embeded in fibrin matrix and fixed in isolation chamber. PHD (prolyl hydroxylases) inhibitor DMOG (Dimethyloxallyl Glycine) was applied systemically in the rats in 40 mg/KG at day 0 and day 3 (DMOG-1), or in 15 mg/KG at day 8, day10 and day12 (DMOG-2). Two weeks later the specimens were explanted and underwent morphological and molecular evaluations.
Compared to the control group, in the DMOG-2 group the HIF-1α positive rate was siginicantly raised as shown in immunohistochemistry staining, accompanied with a smaller cross section area and greater vessel density, and a HIF-1α accumulation in the kidney. The mRNA of HIF-1α and its angiogenic target gene all increased in different extends. Ki67 IHC demostrate more positive cells. There were no significant change in the DMOG-1 group.
By applying DMOG systemically, HIF-1α was up-regulated at the protein level and at the mRNA level, acompanied with angiogenic target gene up-regulateion, and the vascularization was promoted correspondingly. DMOG given at lower dosage constantly after one week tends to have better effect than the group given at larger dosage in the early stage in this model, and promotes cell proliferation, as evidenced by Ki67 IHC. Thus, this study proves that HIF-1a up-regulation is the stimulus for vascularization in the AV loop model and that the process of the vessel outgrowth can be controlled in the AV Loop model utilizing this mechanism.
Electronic supplementary material
The online version of this article (doi:10.1186/s12896-014-0112-x) contains supplementary material, which is available to authorized users.
PMCID: PMC4298964  PMID: 25543909
Hypoxia; Vasculorization; Arteriovenous loop; PHD inhibitor; Dimethyloxallyl Glycine; Kinetics
6.  Protein surface charge of trypsinogen changes its activation pattern 
BMC Biotechnology  2014;14(1):109.
Trypsinogen is the inactive precursor of trypsin, a serine protease that cleaves proteins and peptides after arginine and lysine residues. In this study, human trypsinogen was used as a model protein to study the influence of electrostatic forces on protein–protein interactions. Trypsinogen is active only after its eight-amino-acid-long activation peptide has been cleaved off by another protease, enteropeptidase. Trypsinogen can also be autoactivated without the involvement of enteropeptidase. This autoactivation process can occur if a trypsinogen molecule is activated by another trypsin molecule and therefore is based on a protein–protein interaction.
Based on a rational protein design based on autoactivation-defective guinea pig trypsinogen, several amino acid residues, all located far away from the active site, were changed to modify the surface charge of human trypsinogen. The influence of the surface charge on the activation pattern of trypsinogen was investigated. The autoactivation properties of mutant trypsinogen were characterized in comparison to the recombinant wild-type enzyme. Surface-charged trypsinogen showed practically no autoactivation compared to the wild-type but could still be activated by enteropeptidase to the fully active trypsin. The kinetic parameters of surface-charged trypsinogen were comparable to the recombinant wild-type enzyme.
The variant with a modified surface charge compared to the wild-type enzyme showed a complete different activation pattern. Our study provides an example how directed modification of the protein surface charge can be utilized for the regulation of functional protein–protein interactions, as shown here for human trypsinogen.
Electronic supplementary material
The online version of this article (doi:10.1186/s12896-014-0109-5) contains supplementary material, which is available to authorized users.
PMCID: PMC4299543  PMID: 25543846
Protein design; Protein expression; Protein-interaction; Protein engineering; trypsin
7.  Comparison of growth factor adsorbed scaffold and conventional scaffold with growth factor supplemented media for primary human articular chondrocyte 3D culture 
BMC Biotechnology  2014;14(1):108.
Cartilage tissue engineering offers new strategies in repairing damaged cartilage. Scaffolds have been used for the in vitro and in vivo procedures for this application, which demonstrates the compatible biological and physical properties that mimic natural tissues. Several types of scaffolds were used and had different effects on cell functions. The study was designed to develop a functional gelatin scaffold by adsorption of hyaluronan (HA) and the transforming growth factor β3 (TGF-β3) in a commercially available gelatin scaffold.
The biological properties of human articular chondrocytes were investigated during a 21-day cultivation embedded in either HA + TGF-β3 adsorbed scaffolds or the conventional supplemented method. The rising of proliferation of chondrocytes embedded in adsorbed scaffolds was observed at day 17 and 21 of cultivation (1.27 and 1.28 fold, respectively). The chondrogenic gene expression of the chondrocytes embedded in HA + TGF-β3 adsorbed scaffolds significantly increased: SOX-9 (1.65 fold), ACAN (7.65 fold) and COL2A1 (1.83 fold). Remarkably, over the 21 days of cultivation, HA + TGF-β3 adsorbed scaffolds promoted the extracellular matrix molecules production with higher accumulation of HA (1.2 fold), collagen (1.42 fold) and uronic acid (1.41 fold). Moreover, the cell population and extracellular matrix production, which were examined by a histological analysis and a scanning electron microscope, were correlated with the biochemical analysis.
A small amount of HA and TGF-β3 initially adsorbed in the scaffolds (70 μg and 10 ng, respectively) was consumed over the 21-day cultivation. The HA + TGF-β3 adsorbed gelatin scaffold is effective and more suitable than the conventional supplemented method for the in vitro assessment of human chondrocyte 3D culture.
PMCID: PMC4299815  PMID: 25543823
Gelatin scaffold; Human articular chondrocytes; Extracellular matrix; HA; TGF-β3; Cartilage tissue engineering
8.  Characterization of the substitution pattern of cellulose derivatives using carbohydrate-binding modules 
BMC Biotechnology  2014;14(1):2.
Derivatized celluloses, such as methylcellulose (MC) and hydroxypropyl methylcellulose (HPMC), are of pharmaceutical importance and extensively employed in tablet matrices. Each batch of derivatized cellulose is thoroughly characterized before utilized in tablet formulations as batch-to-batch differences can affect drug release. The substitution pattern of the derivatized cellulose polymers, i.e. the mode on which the substituent groups are dispersed along the cellulose backbone, can vary from batch-to-batch and is a factor that can influence drug release.
In the present study an analytical approach for the characterization of the substitution pattern of derivatized celluloses is presented, which is based on the use of carbohydrate-binding modules (CBMs) and affinity electrophoresis. CBM4-2 from Rhodothermus marinus xylanase 10A is capable of distinguishing between batches of derivatized cellulose with different substitution patterns. This is demonstrated by a higher migration retardation of the CBM in acrylamide gels containing batches of MC and HPMC with a more heterogeneous distribution pattern.
We conclude that CBMs have the potential to characterize the substitution pattern of cellulose derivatives and anticipate that with use of CBMs with a very selective recognition capacity it will be possible to more extensively characterize and standardize important carbohydrates used for instance in tablet formulation.
PMCID: PMC4302574  PMID: 25540113
Substitution pattern; Methylcellulose; Hydroxypropyl methylcellulose; Application of carbohydrate-binding modules
9.  Modifications of cysteine residues in the transmembrane and cytoplasmic domains of a recombinant hemagglutinin protein prevent cross-linked multimer formation and potency loss 
BMC Biotechnology  2014;14(1):1.
Recombinant hemagglutinin (rHA) is the active component in Flublok®; a trivalent influenza vaccine produced using the baculovirus expression vector system (BEVS). HA is a membrane bound homotrimer in the influenza virus envelope, and the purified rHA protein assembles into higher order rosette structures in the final formulation of the vaccine. During purification and storage of the rHA, disulfide mediated cross-linking of the trimers within the rosette occurs and results in reduced potency. Potency is measured by the Single Radial Immuno-diffusion (SRID) assay to determine the amount of HA that has the correct antigenic form.
The five cysteine residues in the transmembrane (TM) and cytoplasmic (CT) domains of the rHA protein from the H3 A/Perth/16/2009 human influenza strain have been substituted to alanine and/or serine residues to produce three different site directed variants (SDVs). These SDVs have been evaluated to determine the impact of the TM and CT cysteines on potency, cross-linking, and the biochemical and biophysical properties of the rHA. Modification of these cysteine residues prevents disulfide bond cross-linking in the TM and CT, and the resulting rHA maintains potency for at least 12 months at 25°C. The strategy of substituting TM and CT cysteines to prevent potency loss has been successfully applied to another H3 rHA protein (from the A/Texas/50/2012 influenza strain) further demonstrating the utility of the approach.
rHA potency can be maintained by preventing non-specific disulfide bonding and cross-linked multimer formation. Substitution of carboxy terminal cysteines is an alternative to using reducing agents, and permits room temperature storage of the vaccine.
PMCID: PMC4320835  PMID: 25540031
Hemagglutinin; Influenza; Vaccine; Potency; Protein cross-linking; Protein stability; Antigen; Cysteine
10.  Comparison of mcl-Poly(3-hydroxyalkanoates) synthesis by different Pseudomonas putida strains from crude glycerol: citrate accumulates at high titer under PHA-producing conditions 
BMC Biotechnology  2014;14(1):962.
Achieving a sustainable society requires, among other things, the use of renewable feedstocks to replace chemicals obtained from petroleum-derived compounds. Crude glycerol synthesized inexpensively as a byproduct of biodiesel production is currently considered a waste product, which can potentially be converted into value-added compounds by bacterial fermentation. This study aimed at evaluating several characterized P. putida strains to produce medium-chain-length poly(3-hydroxyalkanoates) (mcl-PHA) using raw glycerol as the only carbon/energy source.
Among all tested strains, P. putida KT2440 most efficiently synthesized mcl-PHA under nitrogen-limiting conditions, amassing more than 34% of its cell dry weight as PHA. Disruption of the PHA depolymerase gene (phaZ) in P. putida KT2440 enhanced the biopolymer titer up to 47% PHA (%wt/wt). The low biomass and PHA titer found in the mutant strain and the wild-type strain KT2440 seems to be triggered by the high production of the side-product citrate during the fermentation process which shows a high yield of 0.6 g/g.
Overall, this work demonstrates the importance of choosing an appropriate microbe for the synthesis of mcl-PHA from waste materials, and a close inspection of the cell metabolism in order to identify undesired compounds that diminish the availability of precursors in the synthesis of biopolymers such as polyhydroxyalkanoates. Future metabolic engineering works should focus on reducing the production of citrate in order to modulate resource allocation in the cell’s metabolism of P. putida, and finally increase the biopolymer production.
PMCID: PMC4299480  PMID: 25532606
Pseudomonas putida strains; mcl-polyhydroxyalkanoates; Raw glycerol; PHA depolymerase; Metabolic engineering; Citrate
11.  Highly thermostable GH39 β-xylosidase from a Geobacillus sp. strain WSUCF1 
BMC Biotechnology  2014;14(1):963.
Complete enzymatic hydrolysis of xylan to xylose requires the action of endoxylanase and β-xylosidase. β-xylosidases play an important part in hydrolyzing xylo-oligosaccharides to xylose. Thermostable β-xylosidases have been a focus of attention as industrially important enzymes due to their long shelf life and role in the relief of end-product inhibition of xylanases caused by xylo-oligosaccharides. Therefore, a highly thermostable β-xylosidase with high specific activity has significant potential in lignocellulose bioconversion.
A gene encoding a highly thermostable GH39 β-xylosidase was cloned from Geobacillus sp. strain WSUCF1 and expressed in Escherichia coli. Recombinant β-xylosidase was active over a wide range of temperatures and pH with optimum temperature of 70°C and pH 6.5. It exhibited very high thermostability, retaining 50% activity at 70°C after 9 days. WSUCF1 β-xylosidase is more thermostable than β-xylosidases reported from other thermophiles (growth temperature ≤ 70°C). Specific activity was 133 U/mg when incubated with p-nitrophenyl xylopyranoside, with Km and Vmax values of 2.38 mM and 147 U/mg, respectively. SDS-PAGE analysis indicated that the recombinant enzyme had a mass of 58 kDa, but omitting heating prior to electrophoresis increased the apparent mass to 230 kDa, suggesting the enzyme exists as a tetramer. Enzyme exhibited high tolerance to xylose, retained approximately 70% of relative activity at 210 mM xylose concentration. Thin layer chromatography showed that the enzyme had potential to convert xylo-oligomers (xylobiose, triose, tetraose, and pentaose) into fermentable xylose. WSUCF1 β-xylosidase along with WSUCF1 endo-xylanase synergistically converted the xylan into fermentable xylose with more than 90% conversion.
Properties of the WSUCF1 β-xylosidase i.e. high tolerance to elevated temperatures, high specific activity, conversion of xylo-oligomers to xylose, and resistance to inhibition from xylose, make this enzyme potentially suitable for various biotechnological applications.
PMCID: PMC4300165  PMID: 25532585
Lignocellulose; Biofuels; β-xylosidase; Thermostable
12.  Developing a xylanase XYNZG from Plectosphaerella cucumerina for baking by heterologously expressed in Kluyveromyces lactis 
BMC Biotechnology  2014;14(1):107.
Xylanase can replace chemical additives to improve the volume and sensory properties of bread in the baking. Suitable baking xylanase with improved yield will promote the application of xylanase in baking industry. The xylanase XYNZG from the Plectosphaerella cucumerina has been previously characterized by heterologous expression in Pichia pastoris. However, P. pastoris is not a suitable host for xylanase to be used in the baking process since P. pastoris does not have GRAS (Generally Regarded As Safe) status and requires large methanol supplement during the fermentation in most conditions, which is not allowed to be used in the food industry. Kluyveromyces lactis, as another yeast expression host, has a GRAS status, which has been successfully used in food and feed applications. No previous work has been reported concerning the heterologous expression of xylanase gene xynZG in K. lactis with an aim for application in baking.
The xylanase gene xynZG from the P. cucumerina was heterologously expressed in K. lactis. The recombinant protein XYNZG in K. lactis presented an approximately 19 kDa band on SDS-PAGE and zymograms analysis. Transformant with the highest halo on the plate containing the RBB-xylan (Remazol Brilliant Blue-xylan) was selected for the flask fermentation in different media. The results indicated that the highest activity of 115 U/ml at 72 h was obtained with the YLPU medium. The mass spectrometry analysis suggested that the hydrolytic products of xylan by XYNZG were mainly xylobiose and xylotriose. The results of baking trials indicated that the addition of XYNZG could reduce the kneading time of dough, increase the volume of bread, improve the texture, and have more positive effects on the sensory properties of bread.
Xylanase XYNZG is successfully expressed in K. lactis, which exhibits the highest activity among the published reports of the xylanase expression in K. lactis. The recombinant XYNZG can be used to improve the volume and sensory properties of bread. Therefore, the expression yield of recombinant XYNZG can be further improved through engineered strain containing high copy numbers of the XYNZG, and optimized fermentation condition, making bread-baking application possible.
PMCID: PMC4297440  PMID: 25511290
Xylanase; Heterologous expression; Kluyveromyces lactis; Baking
13.  Saccharification of rice straw by cellulase from a local Trichoderma harzianum SNRS3 for biobutanol production 
BMC Biotechnology  2014;14(1):103.
Rice straw has shown to be a promising agricultural by-product in the bioconversion of biomass to value-added products. Hydrolysis of cellulose, a main constituent of lignocellulosic biomass, is a requirement for fermentable sugar production and its subsequent bioconversion to biofuels such as biobutanol. The high cost of commercial enzymes is a major impediment to the industrial application of cellulases. Therefore, the use of local microbial enzymes has been suggested. Trichoderma harzianum strains are potential CMCase and β-glucosidase producers. However, few researches have been reported on cellulase production by T. harzianum and the subsequent use of the crude cellulase for cellulose enzymatic hydrolysis. For cellulose hydrolysis to be efficiently performed, the presence of the whole set of cellulase components including exoglucanase, endoglucanase, and β-glucosidase at a considerable concentration is required. Biomass recalcitrance is also a bottleneck in the bioconversion of agricultural residues to value-added products. An effective pretreatment could be of central significance in the bioconversion of biomass to biofuels.
Rice straw pretreated using various concentrations of NaOH was subjected to enzymatic hydrolysis. The saccharification of rice straw pretreated with 2% (w/v) NaOH using crude cellulase from local T. harzianum SNRS3 resulted in the production of 29.87 g/L reducing sugar and a yield of 0.6 g/g substrate. The use of rice straw hydrolysate as carbon source for biobutanol fermentation by Clostridium acetobutylicum ATCC 824 resulted in an ABE yield, ABE productivity, and biobutanol yield of 0.27 g/g glucose, 0.04 g/L/h and 0.16 g/g glucose, respectively. As a potential β-glucosidase producer, T. harzianum SNRS3 used in this study was able to produce β-glucosidase at the activity of 173.71 U/g substrate. However, for cellulose hydrolysis to be efficient, Filter Paper Activity at a considerable concentration is also required to initiate the hydrolytic reaction. According to the results of our study, FPase is a major component of cellulose hydrolytic enzyme complex system and the reducing sugar rate-limiting enzyme.
Our study revealed that rice straw hydrolysate served as a potential substrate for biobutanol production and FPase is a rate-limiting enzyme in saccharification.
PMCID: PMC4298951  PMID: 25496491
Rice straw; Saccharification; Biobutanol; Trichoderma harzianum SNRS3
14.  The characteristics of bacterial nanocellulose gel releasing silk sericin for facial treatment 
BMC Biotechnology  2014;14(1):104.
Recently, naturally derived facial masks with beneficial biological properties have received increasing interest. In this study, silk sericin-releasing bacterial nanocellulose gel was developed to be applied as a bioactive mask for facial treatment.
The silk sericin-releasing bacterial nanocellulose gel produced at a pH of 4.5 had an ultrafine and extremely pure fiber network structure. The mechanical properties and moisture absorption ability of the gel were improved, compared to those of the commercially available paper mask. Silk sericin could be control-released from the gel. A peel test with porcine skin showed that the gel was less adhesive than the commercially available paper mask, which would be removed from the face more easily without pain. The in vitro cytotoxicity test showed that the gel was not toxic to L929 mouse fibroblast and HaCaT human keratinocyte cells. Furthermore, when implanted subcutaneously and evaluated according to ISO10993-6 standard, the gel was not irritant to tissue.
The silk sericin-releasing bacterial nanocellulose gel had appropriate physical and biological properties and safety for the facial treatment application.
PMCID: PMC4265328  PMID: 25487808
Bacterial nanocellulose; Gluconacetobacter xylinus; Coconut water; Silk sericin; Facial mask
15.  Generation in yeast of recombinant virus-like particles of porcine circovirus type 2 capsid protein and their use for a serologic assay and development of monoclonal antibodies 
BMC Biotechnology  2014;14(1):100.
Porcine circovirus type 2 (PCV2) is considered to be an important emerging pathogen associated with a number of different syndromes and diseases in pigs known as PCV2-associated diseases. It has been responsible for significant mortality among pigs and remains a serious economic problem to the swine industry worldwide leading to significant negative impacts on profitability of pork production.
In this study we have demonstrated that PCV2 capsid (Cap) protein based virus-like particles (VLPs) were efficiently produced in yeast S. cerevisiae and induced production of monoclonal antibodies (MAbs) reactive with virus-infected cells. Moreover, PCV2 Cap VLPs served as a highly specific recombinant antigen for the development of an indirect IgG PCV2 Cap VLP-based ELISA for the detection of virus-specific IgG antibodies in swine sera. Four hundred-nine serum samples collected from pigs in Lithuania were tested for PCV2-specific IgG to determine the sensitivity and specificity of the newly developed ELISA in parallel using a commercial SERELISA test as a gold standard. From 409 tested serum samples, 297 samples were positive by both assays. Thirty-nine sera from 112 serum samples were determined as negative by SERELISA but were found to be positive both in the newly developed indirect IgG PCV2 Cap VLP-based ELISA and the PCR test.
We have demonstrated that S. cerevisiae expression system is an alternative to insect/baculovirus expression system for production of homogenous in size and shape PCV2 Cap protein-based VLPs similar to native virions. Yeast expression system tolerated native virus genes encoding PCV2 Cap protein variants as well as the codon-optimized gene. Moreover, yeast-derived PCV2 Cap VLPs were capable to induce the generation of PCV2-specific MAbs that did not show any cross-reactivity with PCV1-infected cells. The high sensitivity and specificity of the indirect IgG PCV2 Cap VLP-based ELISA clearly suggested that this assay is potentially useful diagnostic tool for screening PCV2–suspected samples.
PMCID: PMC4265424  PMID: 25487652
Virus-like particles; Porcine circovirus 2; Monoclonal antibodies
16.  Characterization of anode and anolyte community growth and the impact of impedance in a microbial fuel cell 
BMC Biotechnology  2014;14(1):102.
A laboratory-scale two-chamber microbial fuel cell employing an aerated cathode with no catalyst was inoculated with mixed inoculum and acetate as the carbon source.
Electrochemical impedance spectroscopy (EIS) was used to study the behavior of the MFC during initial biofilm (week 1) and maximum power density (week 20). EIS were performed on the anode chamber, biofilm (without anolyte) and anolyte (without biofilm). Nyquist plots of the EIS data were fitted with two equivalent electrical circuits to estimate the contributions of intrinsic resistances to the overall internal MFC impedance at weeks 1 and 20, respectively.
The results showed that the system tended to increase power density from 15 ± 3 (week 1) to 100 ± 15 mW/m2 (week 20) and current density 211 ± 7 (week 1) to 347 ± 29 mA/m2 (week 20). The Samples were identified by pyrosequencing of the 16S rRNA gene and showed that initial inoculum (week 1) was constituted by Proteobacteria (40%), Bacteroidetes (22%) and Firmicutes (18%). At week 20, Proteobacterial species were predominant (60%) for electricity generation in the anode biofilm, being 51% Rhodopseudomonas palustris. Meanwhile on anolyte, Firmicutes phylum was predominant with Bacillus sp.
This study proved that under the experimental conditions used there is an important contribution from the interaction of the biofilm and the anolyte on cell performance. Table 1 presents a summary of the specific influence of each element of the system under study.
The results showed certain members of the bacterial electrode community increased in relative abundance from the initial inoculum. For example, Proteobacterial species are important for electricity generation in the anode biofilms and Firmicutes phylum was predominant on anolyte to transfer electron.R1 is the same in the three systems and no variation is observed over time.The biofilm makes a significant contribution to the charge transfer processes at the electrode (R2 and Cdl) and, consequently, on the performance of the anode chamber.The biofilm can act as a barrier which reduces diffusion of the anolyte towards the electrode, all the while behaving like a porous material.The anolyte and its interaction with the biofilm exert a considerable influence on diffusion processes, given that it presents the highest values for Rd which increased at week 20.
PMCID: PMC4299683  PMID: 25487741
Microbial fuel cell; Community growth and electrochemical impedance spectroscopy
17.  Detection of distinct glycosylation patterns on human γ-glutamyl transpeptidase 1 using antibody-lectin sandwich array (ALSA) technology 
BMC Biotechnology  2014;14(1):101.
γ-Glutamyl transpeptidase 1 (GGT1) is an N-glycosylated membrane protein that catabolizes extracellular glutathione and other γ-glutamyl-containing substrates. In a variety of disease states, including tumor formation, the enzyme is shed from the surface of the cell and can be detected in serum. The structures of the N-glycans on human GGT1 (hGGT1) have been shown to be tissue-specific. Tumor-specific changes in the glycans have also been observed, suggesting that the N-glycans on hGGT1 would be an important biomarker for detecting tumors and monitoring their progression during treatment. However, the large quantities of purified protein required to fully characterize the carbohydrate content poses a significant challenge for biomarker development. Herein, we investigated a new antibody-lectin sandwich array (ALSA) platform to determine whether this microanalytical technique could be applied to the characterization of N-glycan content of hGGT1 in complex biological samples.
Our data show that hGGT1 can be isolated from detergent extracted membrane proteins by binding to the ALSA platform. Probing hGGT1 with lectins enables characterization of the N-glycans. We probed hGGT1 from normal human liver tissue, normal human kidney tissue, and hGGT1 expressed in the yeast Pichia pastoris. The lectin binding patterns obtained with the ALSA platform are consistent with the hGGT1 N-glycan composition obtained from previous large-scale hGGT1 N-glycan characterizations from these sources. We also validate the implementation of the Microcystis aeruginosa lectin, microvirin, in this platform and provide refined evidence for its efficacy in specifically recognizing high-mannose-type N-glycans, a class of carbohydrate modification that is distinctive of hGGT1 expressed by many tumors.
Using this microanalytical approach, we provide proof-of-concept for the implementation of ALSA in conducting high-throughput studies aimed at investigating disease-related changes in the glycosylation patterns on hGGT1 with the goal of enhancing clinical diagnoses and targeted treatment regimens.
Electronic supplementary material
The online version of this article (doi:10.1186/s12896-014-0101-0) contains supplementary material, which is available to authorized users.
PMCID: PMC4297448  PMID: 25479762
γ-Glutamyl transpeptidase; Antibody-lectin sandwich arrays; N-glycans
18.  Mesenchymal stem cell spheroids exhibit enhanced in-vitro and in-vivo osteoregenerative potential 
BMC Biotechnology  2014;14(1):105.
Mesenchymal stem cells (MSCs) are a favored cell source for regenerative medicine because of their multilinage potential. However, the conventional monolayer technique used to culture MSCs, inadequately overcomes their low differentiation capacity. Culture of MSCs in multicellular spheroids, more accurately mimics the in-vivo microenvironment; thus, resolving this problem. In this study, we assessed whether the osteoregenerative potential of MSC spheroids is greater than that of monolayer MSCs.
MSC spheroids were generated from rat MSCs (rMSCs) using low-binding plates. Real-time reverse transcription-polymerase chain reaction and immunocytochemical analysis indicated that osteogenic properties were accelerated in MSC spheroids compared with monolayer rMSCs when treated with an osteoblast-inducer reagent for 7 days. Moreover, increased calcium deposition was visualized in MSC spheroids using Alizarin red staining. In a rat calvarial defect model, micro-computed tomography and histological assays showed that MSC spheroid-engrafted defects experienced enhanced bone regeneration.
Our in-vitro and in-vivo results reveal that MSCs in the spheroid culture exhibit enhanced osteoregenerative efficiency compared with monolayer MSCs.
PMCID: PMC4299781  PMID: 25479895
Spheroid; Mesenchymal stem cell; Osteogenesis; Rat calvarial defect; Bone regeneration
19.  Direct analysis of mAb aggregates in mammalian cell culture supernatant 
BMC Biotechnology  2014;14(1):99.
Protein aggregation during monoclonal antibody (mAb) production can occur in upstream and downstream processing (DSP). Current methods to determine aggregate formation during cell culture include size exclusion chromatography (SEC) with a previous affinity chromatography step in order to remove disturbing cell culture components. The pre-purification step itself can already influence protein aggregation and therefore does not necessarily reflect the real aggregate content present in cell culture. To analyze mAb aggregate formation directly in the supernatant of Chinese hamster ovary (CHO) cell culture, we established a protocol, which allows aggregate quantification using SEC, without a falsifying pre-purification step.
The use of a 3 μm silica SEC column or a SEC column tailored for mAb aggregate analysis allows the separation of mAb monomer and aggregates from disturbing cell culture components, which enables aggregate determination directly in the supernatant. Antibody aggregate analysis of a mAb-producing CHO DG44 cell line demonstrated the feasibility of the method. Astonishingly, the supernatant of the CHO cells consisted of over 75% mAb dimer and larger oligomers, representing a substantially higher aggregate content than reported in literature so far.
This study highlights that aggregate quantification directly in the cell culture supernatant using appropriate SEC columns with suitable mAb aggregate standards is feasible without falsification by previous affinity chromatography. Moreover, our results indicate that aggregate formation should be addressed directly in the cell culture and is not only a problem in DSP.
Electronic supplementary material
The online version of this article (doi:10.1186/s12896-014-0099-3) contains supplementary material, which is available to authorized users.
PMCID: PMC4256052  PMID: 25431119
Protein aggregation; Monoclonal antibodies; Mammalian cell culture; CHO cells
20.  Molecular characterization of two A-type P450s, WsCYP98A and WsCYP76A from Withania somnifera (L.) Dunal: expression analysis and withanolide accumulation in response to exogenous elicitations 
BMC Biotechnology  2014;14(1):89.
Pharmacological investigations position withanolides as important bioactive molecules demanding their enhanced production. Therefore, one of the pivotal aims has been to gain knowledge about complete biosynthesis of withanolides in terms of enzymatic and regulatory genes of the pathway. However, the pathway remains elusive at the molecular level. P450s monooxygenases play a crucial role in secondary metabolism and predominantly help in functionalizing molecule core structures including withanolides.
In an endeavor towards identification and characterization of different P450s, we here describe molecular cloning, characterization and expression analysis of two A-type P450s, WsCYP98A and WsCYP76A from Withania somnifera. Full length cDNAs of WsCYP98A and WsCYP76A have open reading frames of 1536 and 1545 bp encoding 511 (58.0 kDa) and 515 (58.7 kDa) amino acid residues, respectively. Entire coding sequences of WsCYP98A and WsCYP76A cDNAs were expressed in Escherichia coli BL21 (DE3) using pGEX4T-2 expression vector. Quantitative real-time PCR analysis indicated that both genes express widely in leaves, stalks, roots, flowers and berries with higher expression levels of WsCYP98A in stalks while WsCYP76A transcript levels were more obvious in roots. Further, transcript profiling after methyl jasmonate, salicylic acid, and gibberellic acid elicitations displayed differential transcriptional regulation of WsCYP98A and WsCYP76A. Copious transcript levels of both P450s correlated positively with the higher production of withanolides.
Two A-types P450 WsCYP98A and WsCYP76A were isolated, sequenced and heterologously expressed in E. coli. Both P450s are spatially regulated at transcript level showing differential tissue specificity. Exogenous elicitors acted as both positive and negative regulators of mRNA transcripts. Methyl jasmonate and salicylic acid resulted in copious expression of WsCYP98A and WsCYP76A. Enhanced mRNA levels also corroborated well with the increased accumulation of withanolides in response to elicitations. The empirical findings suggest that elicitors possibly incite defence or stress responses of the plant by triggering higher accumulation of withanolides.
Electronic supplementary material
The online version of this article (doi:10.1186/s12896-014-0089-5) contains supplementary material, which is available to authorized users.
PMCID: PMC4247701  PMID: 25416924
Withania somnifera; Withanolides; Cytochrome P450 monooxygenase; mRNA; Phytohormones; E.coli
21.  Optimization of production, purification and lyophilisation of cellobiose dehydrogenase by Sclerotium rolfsii 
BMC Biotechnology  2014;14(1):97.
The enzyme cellobiose dehydrogenase (CDH) can be used to oxidize lactose to lactobionic acid. As Sclerotium rolfsii is known to be a good producer of CDH, the aim of this paper was to simplify its production and secondly to systematically study its purification aiming for a high yield. Two preservation methods (freezing and freeze-drying) and the influence of several protectants were investigated.
Production of cellobiose dehydrogenase was optimized leading to a more simplified medium composition. Purification of the enzyme was evaluated by determining breakthrough profiles on different ion exchange (IEX) and hydrophobic interaction (HIC) materials with regard to buffer composition. Highest purification with an acceptable loss during the capture step using IEX was obtained with a Q Sepharose XL medium and a 100 mM sodium acetate buffer at pH 4.5. Subsequent purification using hydrophobic interaction chromatography was done at 1.1 M ammonium sulfate concentration. Purification was moderate, yielding a specific activity of 11.9 U/mg (56% yield). However, as could be shown in a preliminary experiment, purity of the obtained enzyme solution was sufficient for its intended use to oxidize lactose to lactobionic acid. Various sugars and sugar alcohols were investigated to study their protective effect during lyophilisation and freezing at -20°C. Glucose and lactulose could be identified to have a high lyoprotective effect while loss of enzyme activity was high (77%) when using no additives.
By simplifying the cultivation medium of Sclerotium rolfsii, the costs of cellobiose dehydrogenase production could be reduced. Simultaneously, CDH production was increased by 21%. The production of lactobionic acid from lactose is possible using partially purified and unpurified enzyme. Storage at -20°C using 50% (w/v) glycerol was considered to be most suited for preservation of the enzyme.
PMCID: PMC4241217  PMID: 25407159
Cellobiose dehydrogenase; CDH; Sclerotium rolfsii; Enzyme purification; Lyophilisation; Cryoprotection; Lactobionic acid
22.  Development of an enzyme linked immunosorbent assay for detection of cyathane diterpenoids 
BMC Biotechnology  2014;14(1):98.
So-called cyathane type diterpenoids are produced as secondary metabolites by basidiomycetes. Based on their antibacterial, fungicidal, and cytotoxic properties, cyathane type terpenoids represent interesting target compounds in fungal biotechnology.
An indirect competitive enzyme linked immunosorbent assay has been developed for detection of cyathane type diterpenoids. Rabbit polyclonal antibodies were raised against a mixture of striatal A and B conjugated to bovine serum albumin. The conditions for direct attachment of the hapten striatal B to a solid phase by passive adsorption were optimized. The cross reactivities of the striatals A, C and D, of the striatins A and B, and of the erinacines C and P to striatal B were determined. The validation study showed that the ELISA was precise and sensitive. The average IC50 of striatal B was 36.0 ng mL−1 with an inter-assay coefficient of variation (CV) of 13.2% (n = 5). Recoveries from striatal B spiked samples in the assay were in the range of 97.3 – 125.9%. A good correlation between the striatal B concentration measured by the ELISA and by HPLC-DAD (y = 1.1122× – 0.1585, R2 = 0.9942) was obtained from linear regression analysis. The suitability of the ELISA for detection of cyathane type diterpenoids in submerged cultures and fruiting bodies of H. erinaceus was studied. It showed cross reactivity with supernatants from submerged cultures and extracts thereof, but did not show cross reactivity with extracts from fruiting bodies.
The developed method is appropriate for qualitative and quantitative detection of cyathane diterpenoids in complex mixtures. Due to its high sensitivity and specificity, it represents an ideal screening method for discovering new cyathane diterpenoids and new potential producers of them.
Electronic supplementary material
The online version of this article (doi:10.1186/s12896-014-0098-4) contains supplementary material, which is available to authorized users.
PMCID: PMC4239385  PMID: 25404227
Cyathane diterpenoids; ELISA; Hericium erinaceus; Striatal; Erinacine
23.  Targeted gene disruption by use of transcription activator-like effector nuclease (TALEN) in the water flea Daphnia pulex 
BMC Biotechnology  2014;14(1):95.
The cosmopolitan microcrustacean Daphnia pulex provides a model system for both human health research and monitoring ecosystem integrity. It is the first crustacean to have its complete genome sequenced, an unprecedented ca. 36% of which has no known homologs with any other species. Moreover, D. pulex is ideally suited for experimental manipulation because of its short reproductive cycle, large numbers of offspring, synchronization of oocyte maturation, and other life history characteristics. However, existing gene manipulation techniques are insufficient to accurately define gene functions. Although our previous investigations developed an RNA interference (RNAi) system in D. pulex, the possible time period of functional analysis was limited because the effectiveness of RNAi is transient. Thus, in this study, we developed a genome editing system for D. pulex by first microinjecting transcription activator-like effector nuclease (TALEN) mRNAs into early embryos and then evaluating TALEN activity and mutation phenotypes.
We assembled a TALEN construct specific to the Distal-less gene (Dll), which is a homeobox transcription factor essential for distal limb development in invertebrates and vertebrates, and evaluated its activity in vitro by single-strand annealing assay. Then, we injected TALEN mRNAs into eggs within 1 hour post-ovulation. Injected embryos presented with defects in the second antenna and altered appendage development, and indel mutations were detected in Dll loci, indicating that this technique successfully knocked out the target gene.
We succeeded, for the first time in D. pulex, in targeted mutagenesis by use of Platinum TALENs. This genome editing technique makes it possible to conduct reverse genetic analysis in D. pulex, making this species an even more appropriate model organism for environmental, evolutionary, and developmental genomics.
Electronic supplementary material
The online version of this article (doi:10.1186/s12896-014-0095-7) contains supplementary material, which is available to authorized users.
PMCID: PMC4239399  PMID: 25404042
Daphnia pulex; Distal-less; Platinum TALEN; Gene disruption; Knock-out; Targeted mutagenesis; Gene manipulation; Genome editing
24.  Morphology and ploidy level determination of Pteris vittata callus during induction and regeneration 
BMC Biotechnology  2014;14(1):96.
Morphological and ploidy changes of the arsenic hyperaccumulator, Chinese brake fern (Pteris vittata) callus tissue are described here to provide insight into fern life cycle biology and for possible biotechnology applications. Pteris vittata callus was studied using transmission and scanning electron microscopy, and flow cytometry.
Callus induction occurred both in light and dark culture conditions from prothallus tissues, whereas rhizoid formation occurred only in dark culture conditions. Callus tissues contained two types of cells: one actively dividing and the other containing a single large vacuole undergoing exocytosis. Sporophytes regenerated from callus asynchronously form clusters of cells in a manner apparently analogous to direct organogenesis. Extracellular matrices were observed in actively-growing callus and at the base of regenerating sporophytes. Callus tissue nuclei were found to be primarily diploid at induction and throughout maintenance of cultures indicating that callus cell fate is determined at induction, which closely follows apogamous sporophyte development. Presence of a dense extracellular matrix in conjunction with sporophyte development suggests a link between the suspensor-like activity of the embryonic foot during normal fern embryo development and the suspected functions of extracellular matrices in angiosperms.
Further investigation could lead to a better understanding of genes involved in P. vittata embryo development and apogamous sporophyte development. The methodology could be useful for in vitro propagation of rare and valuable fern germplasm.
Electronic supplementary material
The online version of this article (doi:10.1186/s12896-014-0096-6) contains supplementary material, which is available to authorized users.
PMCID: PMC4241211  PMID: 25404146
Extracellular matrix; Phytoremediation; Chinese brake fern; Pteris vittata; Scanning electron microscopy; Tissue culture and transformation; Transmission electron microscopy
25.  Codon optimization and factorial screening for enhanced soluble expression of human ciliary neurotrophic factor in Escherichia coli 
BMC Biotechnology  2014;14(1):92.
Neurotrophic factors influence survival, differentiation, proliferation and death of neuronal cells within the central nervous system. Human ciliary neurotrophic factor (hCNTF) has neuroprotective properties and is also known to influence energy balance. Consequently, hCNTF has potential therapeutic applications in neurodegenerative, obesity and diabetes related disorders. Clinical and biological applications of hCNTF necessitate a recombinant expression system to produce large amounts of functional protein in soluble form. Earlier attempts to express hCNTF in Escherichia coli (E. coli) were limited by low amounts and the need to refold from inclusion bodies.
In this report, we describe a strategy to effectively identify constructs and conditions for soluble expression of hCNTF in E. coli. Small-scale expression screening with soluble fusion tags identified many conditions that yielded soluble expression. Codon optimized 6-His-hCNTF construct showed soluble expression in all the conditions tested. Large-scale culture of the 6-His-hCNTF construct yielded high (10 – 20 fold) soluble expression (8 – 9 fold) as compared to earlier published reports. Functional activity of recombinant 6-His-hCNTF produced was confirmed by its binding to hCNTF receptor (hCNTFRα) with an EC50 = 36 nM.
Our results highlight the combination of codon optimization and screening soluble fusion tags as a successful strategy for high yielding soluble expression of hCNTF in E. coli. Codon optimization of the hCNTF sequence seems to be sufficient for soluble expression of hCNTF. The combined approach of codon optimization and soluble fusion tag screen can be an effective strategy for soluble expression of pharmaceutical proteins in E. coli.
Electronic supplementary material
The online version of this article (doi:10.1186/s12896-014-0092-x) contains supplementary material, which is available to authorized users.
PMCID: PMC4237735  PMID: 25394427
Neurotrophic factors; Human CNTF; Codon optimization; Recombinant soluble expression; E. coli

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