http://www.biomedcentral.com/bmcbiotechnol/ The latest research articles published by BMC Biotechnology 2009-07-10T00:00:00Z This is an RSS newsfeed from BioMed Central It is intended to be used with an RSS reader. For more information about RSS newsfeeds from BioMed Central, visit http://www.biomedcentral.com/info/about/rss/ Background: Green algae of the family Volvocaceae are a model lineage for studying the molecular evolution of multicellularity and cellular differentiation. The volvocine alga Gonium is intermediate in organizational complexity between its unicellular relative, Chlamydomonas, and its multicellular relatives with differentiated cell types, such as Volvox. Gonium pectorale consists of ~16 biflagellate cells arranged in a flat plate. The detailed molecular analysis of any species necessitates its accessibility to genetic manipulation, but, in volvocine algae, transformation procedures have so far only been established for Chlamydomonas reinhardtii and Volvox carteri. Results: Stable nuclear transformation of G. pectorale was achieved using a heterologous dominant antibiotic resistance gene, the aminoglycoside 3'-phosphotransferase VIII gene (aphVIII) of Streptomyces rimosus, as a selectable marker. Heterologous 3'- and 5'-untranslated flanking sequences, including promoters, were from Chlamydomonas reinhardtii or from Volvox carteri. After particle gun bombardment of wild type Gonium cells with plasmid-coated gold particles, transformants were recovered. The transformants were able to grow in the presence of the antibiotic paromomycin and produced a detectable level of the AphVIII protein. The plasmids integrated into the genome, and stable integration was verified after propagation for over 1400 colony generations. Co-transformants were recovered with a frequency of ~30-50% when cells were co-bombarded with aphVIII-based selectable marker plasmids along with unselectable plasmids containing heterologous genes. The transcription of the co-transformed, unselectable genes was confirmed. After heterologous expression of the luciferase gene from the marine copepod Gaussia princeps, which was previously engineered to match the codon usage in C. reinhardtii, Gonium transformants show luciferase activity through light emission in bioluminescence assays. Conclusion: Flanking sequences that include promoters from C. reinhardtii and from V. carteri work in G. pectorale and allow the functional expression of heterologous genes, such as the selectable marker gene aphVIII of S. rimosus or the co-transformed, codon-optimized G. princeps luciferase gene, which turned out to be a suitable reporter gene in Gonium. The availability of a method for transformation of Gonium makes genetic engineering of this species possible and allows for detailed studies in molecular evolution using the unicellular Chlamydomonas, the 16-celled Gonium, and the multicellular Volvox. http://www.biomedcentral.com/1472-6750/9/64 Kai Lerche Armin Hallmann BMC Biotechnology 2009, 9:64 2009-07-10T00:00:00Z doi:10.1186/1472-6750-9-64 BMC Biotechnology 1472-6750 9 64 2009-07-10T00:00:00Z PDF Background: Double-tagged gene fusions were constructed to overexpress N-acetyl-D-glucosamine 2-epimerase (GlcNAc 2-epimerase) and N-acetyl-D-neuraminic acid aldolase (Neu5Ac aldolase), two sequential enzymes in the production of sialic acids. The fusion proteins GlcNAc 2-epimerase and Neu5Ac aldolase were both tagged with glutathione S-transferase (GST) at the N-terminus, but respectively with five contiguous aspartate residues (5D) and five contiguous arginine residues (5R) at the C-terminus. Results: Both fusion proteins possessing the enzymatic activity could be induced to overexpress in Escherichia coli. These double-tagged fusions were designed such that the surface of expressed fused proteins was charged under the conditions of enzyme reactions. Simply capturing these two fusion proteins by respectively using anionic and cationic exchangers (Sepharose Q and Sepharose SP) through electrostatic binding to 5D and 5R tags allowed the expressed proteins to be isolated and immobilized in a single step. The introduction of double tags could only marginally alter the affinity of these enzymes for their substrates, and the double-tagged proteins were enzymatic active in both soluble and immobilized forms. A combined use of these fusion proteins led to the production of N-acetyl-D-neuraminic acid (Neu5Ac) from N-acetyl-D-glucosamine (GlcNAc). Conclusions: Double-tagged gene fusions were proposed for over-expressing two sequential enzyme proteins, which could be easily immobilzed onto ionic exchangers by direct capture from the crude prtoein extracts via ionic tags. The immobilized, double-tagged proteins were demonstrated to be effective for one-pot enzymatic production of sialic acid. http://www.biomedcentral.com/1472-6750/9/63 Tzu-Hsien Wang Ying-Yin Chen Hsin-Hung Pan Feng-Pao Wang Chung-Hsien Cheng Wen-Chien Lee BMC Biotechnology 2009, 9:63 2009-07-09T00:00:00Z doi:10.1186/1472-6750-9-63 BMC Biotechnology 1472-6750 9 63 2009-07-09T00:00:00Z PDF Background: Platinum nanomaterial is one of the significant noble metal catalysts, and the interaction of platinum with microbe is one of the key factors in influencing the size and the distribution of the platinum nanoparticles on the microbial biomass. Some properties of Pt() adsorption and reduction by resting cells of Bacillus megatherium D01 biomass have once been investigated, still the mechanism active in the platinum biosorption remains to be seen and requires further elucidating.Result: A further insight into the biosorption mechanism of Pt() onto resting cells of Bacillus megatherium D02 biomass on a molecular level has been obtained. The image of scanning electron microscopy (SEM) of the D02 biomass challenged with Pt() displayed a clear distribution of bioreduced platinum particles with sizes of nanometer scale on the biomass. The state of the bioreduction of the Pt() to elemental Pt(0) examined via X-ray photoelectron spectroscopy (XPS) suggested that the biomass reduces the Pt() to Pt() followed by a slower reduction to Pt(0). The analysis of glucose content in the hydrolysates of D02 biomass for different time intervals using ultraviolet-visible (UV-vis) spectrophotometry indicated that certain reducing sugars occur in the hydrolyzed biomass and that the hydrolysis of polysaccharides of the biomass is a rapid process. The infrared (IR) spectrometry on D02 biomass and that challenged with Pt(), and on glucose and that reacted with Pt() demonstrated that the interaction of the biomass with Pt() seems to be through oxygenous or nitrogenous chemical functional groups on the cell wall biopolymers; that the potential binding sites for Pt species include hydroxyl of saccharides, carboxylate anion and carboxyl of amino acid residues, peptide bond, etc.; and that the free monosaccharic group bearing hemiacetalic hydroxyl from the hydrolyzed biomass behaving as an electron donor, in situ reduces the Pt() to Pt(0). And moreover, the binding of the Pt() to the oxygen of the carbonyl group of peptide bond caused a change in the secondary structure of proteins; i.e. a transformation, in polypeptide chains, of b-folded to a-helical form in that the latter might be expected to be more advantageous than b-folded form to the platinum nanoparticles under shelter from gathering although the both special conformations of proteins could be much probably responsible for the stabilization of the particles. Conclusion: That knowledge could serve as a guide in the researches for improving the preparation of highly dispersive supported platinum catalyst and for fabricating new advanced platinum nanostructured devices by biotechnological methods. http://www.biomedcentral.com/1472-6750/9/62 Zhongyu Lin Ru Xue Yiwen Ye Jianhong Zheng Zhenling Xu BMC Biotechnology 2009, 9:62 2009-07-06T00:00:00Z doi:10.1186/1472-6750-9-62 BMC Biotechnology 1472-6750 9 62 2009-07-06T00:00:00Z PDF Background: In protein engineering, site-directed mutagenesis methods are used to generate DNA sequences with mutated codons, insertions or deletions. In a widely used method, mutations are generated by PCR using a pair of oligonucleotide primers designed with mismatching nucleotides at the center of the primers. In this method, primer-primer annealing may prevent cloning of mutant cDNAs. To circumvent this problem we developed an alternative procedure that does not use forward-reverse primer pair in the same reaction. Results: In initial studies we used a double-primer PCR mutagenesis protocol, but sequencing of products showed tandem repeats of primer in cloned DNA. We developed an alternative method that starts with two Single-Primer Reactions IN Parallel using high-fidelity Pwo DNA polymerase. Thus, we call the method with the acronym SPRINP. The SPRINP reactions are then combined, denatured at 95C, and slowly cooled, promoting random annealing of the parental DNA and the newly synthesized strands. The products are digested with DpnI that digests methylated parental strands, and then transformed into E. coli. Using this method we generated >40 mutants in cDNAs coding for human Epithelial Na+ Channel (ENaC) subunits. The method has been tested for 1-3 bp codon mutation and insertion of a 27 bp epitope tag into cDNAs. Conclusion: The SPRINP mutagenesis protocol yields mutants reliably and with high fidelity. The use of a single primer in each amplification reaction increases the probability of success of primers relative to previous methods employing a forward and reverse primer pair in the same reaction. http://www.biomedcentral.com/1472-6750/9/61 Oded Edelheit Aaron Hanukoglu Israel Hanukoglu BMC Biotechnology 2009, 9:61 2009-06-30T00:00:00Z doi:10.1186/1472-6750-9-61 BMC Biotechnology 1472-6750 9 61 2009-06-30T00:00:00Z PDF Background: Ricin is a lethal toxin that inhibits protein synthesis. It is easily extracted from a ubiquitously grown plant, Ricinus communis, and thus readily available for use as a bioweapon (BW). Anti-ricin antibodies provide the only known therapeutic against ricin intoxication. Results: In this study, after immunizing a non-human primate (Macaca fascicularis) with the ricin chain A (RCA), a phage-displayed immune library was built (2x10E8 clones), that included the lambda light chain fragment. The library was screened against ricin, and specific binders were sequenced and further analyzed. The best clone, 43RCA, was isolated using a new, stringent neutralization test. 43RCA had a high, picomolar affinity (41 pM) and neutralized ricin efficiently (CI50= 23+/-3 ng/ml, corresponding to a [scFv]/[ricin] molar ratio of 4). The neutralization capacity of 43RCA compared favourably with that of polyclonal anti-deglycosylated A chain (anti-dgRCA) IgGs, obtained from hyperimmune mouse serum, which were more efficient than any monoclonal at our disposal. The 43RCA sequence is very similar to that for human IgG germline genes, with 162 of 180 identical amino acids for the VH and VL (90% sequence identity). Conclusion: Results of the characterization studies, and the high degree of identity with human germline genes, altogether make this anti-ricin scFv, or an IgG derived from it, a likely candidate for use in humans to minimize effects caused by ricin intoxication. http://www.biomedcentral.com/1472-6750/9/60 Thibaut Pelat Michael Hust Martha Hale Marie-Paule Lefranc Stefan Dubel Philippe Thullier BMC Biotechnology 2009, 9:60 2009-06-30T00:00:00Z doi:10.1186/1472-6750-9-60 BMC Biotechnology 1472-6750 9 60 2009-06-30T00:00:00Z PDF Background: The investigation of molecular mechanisms underlying transcriptional regulation, particularly in embryonic stem cells, has received increasing attention and involves the systematic identification of target genes and the analysis of promoter co-occupancy. High-throughput approaches based on chromatin immunoprecipitation (ChIP) have been widely used for this purpose. However, these approaches remain time-consuming, expensive, labor-intensive, involve multiple steps, and require complex statistical analysis. Advances in this field will greatly benefit from the development and use of simple, fast, sensitive and straightforward ChIP assay and analysis methodologies. Results: We initially developed a simplified, basic ChIP protocol that combines simplicity, speed and sensitivity. ChIP analysis by real-time PCR was compared to analysis by densitometry with the ImageJ software. This protocol allowed the rapid identification of known target genes for SOX2, NANOG, OCT3/4, SOX17, KLF4, RUNX2, OLIG2, SMAD2/3, BMI-1, and c-MYC in a human embryonic stem cell line. We then developed a novel Sequential ChIP protocol to investigate in vivo promoter co-occupancy, which is basically characterized by the absence of antibody-antigen disruption during the assay. It combines centrifugation of agarose beads and magnetic separation. Using this Sequential ChIP protocol we found that c-MYC associates with the SOX2/NANOG/OCT3/4 complex and identified a novel RUNX2/BMI-1/SMAD2/3 complex in BG01V cells. These two TF complexes associate with two distinct sets of target genes. The RUNX2/BMI-1/SMAD2/3 complex is associated predominantly with genes not expressed in undifferentiated BG01V cells, consistent with the reported role of those TFs as transcriptional repressors. Conclusion: These simplified basic ChIP and novel Sequential ChIP protocols were successfully tested with a variety of antibodies with human embryonic stem cells, generated a number of novel observations for future studies and might be useful for high-throughput ChIP-based assays. http://www.biomedcentral.com/1472-6750/9/59 Ricardo Medeiros Kate Papenfuss Brian Hoium Kristen Coley Joy Jadrich Saik-Kia Goh Anuratha Elayaperumal Julio Herrera Ernesto Resnik Hsiao-Tzu Ni BMC Biotechnology 2009, 9:59 2009-06-29T00:00:00Z doi:10.1186/1472-6750-9-59 BMC Biotechnology 1472-6750 9 59 2009-06-29T00:00:00Z PDF Background: Cell-free protein synthesis (CFPS) is a rapid and high throughput technology for obtaining proteins from their genes. The primary energy source ATP is regenerated from the secondary energy source through substrate phosphorylation in CFPS. Results: Distinct from common secondary energy sources (e.g., phosphoenolpyruvate - PEP, glucose-6-phosphate), maltodextrin was used for energizing CFPS through substrate phosphorylation and the glycolytic pathway because (i) maltodextrin can be slowly catabolized by maltodextrin phosphorylase for continuous ATP regeneration, (ii) maltodextrin phosphorylation can recycle one phosphate per reaction for glucose-1-phosphate generation, and (iii) the maltodextrin chain-shortening reaction can produce one ATP per glucose equivalent more than glucose can. Three model proteins, esterase 2 from Alicyclobacillus acidocaldarius, green fluorescent protein, and xylose reductase from Neurospora crassa were synthesized for demonstration. Conclusions: Slowly-metabolized maltodextrin used as a low-cost secondary energy compound for CFPS produced higher levels of proteins than PEP, glucose, and glucose-6-phospahte. The enhancement of protein synthesis was largely attributed to better-controlled phosphate levels (recycling of inorganic phosphate) and a more homeostatic reaction environment. http://www.biomedcentral.com/1472-6750/9/58 Yiran Wang Y-H. Percival Zhang BMC Biotechnology 2009, 9:58 2009-06-28T00:00:00Z doi:10.1186/1472-6750-9-58 BMC Biotechnology 1472-6750 9 58 2009-06-28T00:00:00Z PDF Background: Short interfering RNAs (siRNAs) have become the research tool of choice for gene suppression, with human clinical trials ongoing. The emphasis so far in siRNA therapeutics has been the design of one siRNA with complete complementarity to the intended target. However, there is a need for multi-targeting interfering RNA in diseases in which multiple gene products are of importance. We have investigated the possibility of using a single short synthetic duplex RNA to suppress the expression of VEGF-A and ICAM-1; genes implicated in the progression of ocular neovascular diseases such as diabetic retinopathy. Results: Duplex RNA were designed to have incomplete complementarity with the 3'UTR sequences of both target genes. One such duplex, CODEMIR-1, was found to suppress VEGF and ICAM-1 by 90 and 60%, respectively in ARPE-19 cells at a transfected concentration of 40 ng/mL. Use of a cyan fusion reporter with target sites constructed in its 3'UTR demonstrated that the repression of VEGF and ICAM-1 by CODEMIR-1 was indeed due to interaction with the target sequence. An exhaustive analysis of sequence variants of CODEMIR-1 demonstrated a clear positive correlation between activity against VEGF (but not ICAM-1) and the length of the contiguous complementary region (from the 5' end of the guide strand). Various strategies, including the use of inosine bases at the sites of divergence of the target sequences were investigated. Conclusion: Our work demonstrates the possibility of designing multitargeting dsRNA to suppress more than one disease-altering gene. This warrants further investigation as a possible therapeutic approach. http://www.biomedcentral.com/1472-6750/9/57 Toby Passioura Mary Gozar Amber Goodchild Andrew King Greg Arndt Michael Poidinger Donald Birkett Laurent Rivory BMC Biotechnology 2009, 9:57 2009-06-16T00:00:00Z doi:10.1186/1472-6750-9-57 BMC Biotechnology 1472-6750 9 57 2009-06-16T00:00:00Z XML Background: In recent years, xylanases have attracted considerable research interest because of their potential in various industrial applications. The yeast Pichia pastoris can neither utilize nor degrade xylan, but it possesses many attributes that render it an attractive host for the expression and production of industrial enzymes. Results: The Xyn2 gene, which encodes the main Trichoderma reesei Rut C-30 endo-β-1, 4-xylanase was cloned into the pPICZαA vector and expressed in Pichia pastoris. The selected P. pastoris strains produced as 4,350 nkat/ml β-xylanase under the control of the methanol inducible alcohol oxidase 1 (AOX1) promoter. The secreted recombinant Xyn2 was estimated by SDS-PAGE to be 21 kDa. The activity of the recombinant Xyn2 was highest at 60°C and it was active over a broad range of pH (3.0–8.0) with maximal activity at pH 6.0. The enzyme was quite stable at 50°C and retained more than 94% of its activity after 30 mins incubation at this temperature. Using Birchwood xylan, the determined apparent Km and kcat values were 2.1 mg/ml and 219.2 S-1, respectively. The enzyme was highly specific towards xylan and analysis of xylan hydrolysis products confirmed as expected that the enzyme functions as endo-xylanase with xylotriose as the main hydrolysis products. The produced xylanase was practically free of cellulolytic activity. Conclusion: The P. pastoris expression system allows a high level expression of xylanases. Xylanase was the main protein species in the culture supernatant, and the functional tests indicated that even the non-purified enzyme shows highly specific xylanase activity that is free of cellulolytic side acitivities. Therefore, P pastoris is a very useful expression system when the goal is highly specific and large scale production of glycosyl hydrolases. http://www.biomedcentral.com/1472-6750/9/56 Jun He Bing Yu Keying Zhang Xuemei Ding Daiwen Chen BMC Biotechnology 2009, 9:56 2009-06-16T00:00:00Z doi:10.1186/1472-6750-9-56 BMC Biotechnology 1472-6750 9 56 2009-06-16T00:00:00Z XML Background: Budded baculoviruses are utilized for vaccine, the production of antibody and functional analysis of transmembrane proteins. In this study, we tried to produce and purify the recombinant Bombyx mori nucleopolyhedrovirus (rBmNPV-hPRR) that displayed human (pro)renin receptor (hPRR) connected with FLAG peptide sequence on its own surface. These particles were used for further binding analysis of hPRR to human prorenin. The rBmNPV-hPRR was produced in silkworm larvae and purified from its hemolymph using size exclusion chromatography (SEC). Results: A rapid method of BmNPV titer determination in hemolymph was performed using quantitative real-time PCR (Q-PCR). A correlation coefficient of BmNPV determination between end-point dilution and Q-PCR methods was found to be 0.99. rBmNPV-hPRR bacmid-injected silkworm larvae produced recombinant baculovirus of 1.31 × 108 plaque forming unit (pfu) in hemolymph, which was 2.8 × 104 times higher than transfection solution in Bm5 cells. Its purification yield by Sephacryl S-1000 SF column chromatography was 264 fold from larval hemolymph at 4 days post-injection (p.i.), but 35 or 39 fold at 4.5 or 5 days p.i., respectively. Protein patterns of rBmNPV-hPRR purified at 4 and 5 days were the same and ratio of envelope proteins (76, 45 and 35 kDa) to VP39, one of nucleocapsid proteins, increased at 5 days p.i. hPRR was detected in only purified rBmNPV-hPRR at 5 days p.i.. Conclusion: The successful purification of rBmNPV-hPRR indicates that baculovirus production using silkworm larvae and its purification from hemolymph by Sephacryl S-1000 SF column chromatography can provide an economical approach in obtaining the purified BmNPV stocks with high titer for large-scale production of hPRR. Also, it can be utilized for further binding analysis and screening of inhibitors of hPRR. http://www.biomedcentral.com/1472-6750/9/55 Tatsuya Kato Suganthi Manohar Shigeyasu Tanaka Enoch Park BMC Biotechnology 2009, 9:55 2009-06-12T00:00:00Z doi:10.1186/1472-6750-9-55 BMC Biotechnology 1472-6750 9 55 2009-06-12T00:00:00Z XML