Characterization of a novel bioflocculant, p-KG03, from a marine dinoflagellate, Gyrodinium impudicum KG03

The flocculating activity of an exopolysaccharide, p-KG03, produced by a marine dinoflagellate Gyrodinium impudicum KG03 was investigated. The p-KG03 was a highly sulfated exopolysaccharide that showed strong antiviral activity against encephalomyocarditis virus (EMCV) and immunostimulating activity by NK cell activation. For the industrial applications of p-KG03, as the bioflocculant agent, p-KG03 showed that more than 90% of the flocculating activity in kaolin suspension occurred at concentrations of 0.5 mg/l with the maximum at 1.0 mg/l. However, flocculation decreased from 2.5 mg/l. The flocculation rate increased linearly with concentration and was higher than that observed in commercial products such as polyacrylamide (approximately 1.0 mg/l) or zooglan (approximately 3.0 mg/l). The p-KG03 was an effective flocculant under acidic conditions (pH 3-6) and over a wide temperature range (4-90 degrees C). The presence of cations did not enhance flocculating activity. The average molecular mass, as determined by gel filtration chromatography, was about 1.87 x 10(3) KDa. Galactose was the main sugar in p-KG03, which also contained uronic acid (2.9%, w/w) and sulfate groups (10.3%, w/w). The infrared spectrum of p-KG03 showed absorption bands of carboxylate groups. Thermogravimetric analysis indicated a degradation temperature (T(d)) of 250 degrees C. Several other properties of p-KG03 such as intrinsic viscosity, the rheological behavior, consistency index (k) and flow behavior index (eta) were also studied.     

Screening of a specific monoclonal antibody against and detection ofListeria monocytogenes whole cells using a surface plasmon resonance biosensor

In this study, a specific monoclonal antibody againstListeria monocytogenes was screened using an SPR biosensor Monoclonal antibodies were bound to protein L, after which theL. monocytogenes cells were subjected to an affinity assay. Protein L was immobilized on a carboxymethyl dextran (CM-Dex) surface via an amine coupling method and utilized repeatedly by regeneration. The monoclonal antibody, ‘A18’, was selected and employed for the high-sensitivity detection ofL. monocytogenes. Under optimized conditions, 10³ cells/ml or 50 cells were detected by the SPR biosensor.     

Real-time monitoring of cell-free protein synthesis on a surface plasmon resonance chip

Taking advantage of the "open" nature of cell-free protein synthesis, this study investigated the direct analysis of protein expression using a surface plasmon resonance sensor. During the on-chip incubation of the reaction mixture for cell-free protein synthesis, the expressed protein molecules were immobilized onto the surface of the chip, giving rise to a sensorgram signal, which enabled on-line monitoring of protein expression. In addition, we found that the expression of the aggregation-prone proteins could be effectively monitored. The ability to monitor these proteins was most likely through the instant isolation of the expressed protein molecules onto the solid surface of the chip.     

Dynamic Control of Oligosaccharide Modification in the Mammary Gland: Linking Recombinant Human Erythropoietin

We analyzed two transgenic mouse lines that secrete rhEPO in their milk to assess the dynamic control of N-linked oligosaccharides. Since pharmaceutically available epoetin α and β are produced in CHO cells, we compared transgenic mammary gland-derived rhEPO to its CHO cell-derived counterpart. The major glycosyltransferases that determine the N-oligosaccharides patterns of rhEPO include N-acetylglycosaminyltransferase (GnT) and α1,3/4 fucosyltransferase (Fuc-TIV), GnT-III, -V and Fuc-TIV expression in the mouse mammary gland is significantly higher than that in Chinese hamster ovary (CHO)-derived cells, where the protein is not detectable. The data suggest that N-linked sugar chain patterns of recombinant glycoproteins, produced by the mammary gland differ, since GnT-III alters the sugar pattern extensively. In our experiments, rhEPO produced by the transgenic mice contains more tetra-acidic oligosaccharide structures than epoetin α derived from CHO cells, a rhEPO that is widely used therapeutically. Accordingly, we examined milk-derived rhEPO activity, both in vitro and in vivo. The rhEPO protein purified from the milk of mammary glands upregulates the EPO receptor-mediated expression of the STAT5 gene in MCF-7 cells in a dose-dependent manner, similar to the effects of epoetin α. Furthermore, direct injection of rhEPO into the mouse tail vein leads to an increase in the levels of blood components, such as red blood cells and platelets. In light of these findings, we suggest that the mammary glands of transgenic animals provide a sufficient environment to generate rhEPO with post-translational modifications for biopharmaceutical use. These authors are equal contributors to this work.     

A new approach for discovering cold-active enzymes in a cell mixture of pure-cultured bacteria

To overcome the intrinsic problems of conventional approaches, such as the unavailability of source microorganisms in metagenomic libraries and the production of inactive aggregates, a new method was tested for discovering new enzymes (e.g. cold-active chitinase). A metagenome-like library was constructed using genomes extracted from a cell mixture of pure-cultured chitinolytic bacteria, followed by activity-based screening for Escherichia coli clones that exhibit chitinase activity on selective medium. Within one positive chitinolytic clone, one chitinase gene (chi22718_III) was detected and assigned to the arctic marine bacterium, Pseudoalteromonas issachenkonii PAMC 22718, by colony-PCR with chi22718_III-specific primers. When expressed in E. coli, recombinant R-Chi22718_III lost 85 % of its enzyme activity when pre-incubated at 40 °C for 1 h, whereas its mesophilic counterpart R-ChiK only lost 10 % of its activity under the same conditions indicating that R-Chi22718_III is thermolabile, a characteristic of cold-active enzymes.     

Development of patatin knockdown potato tubers using RNA interference (RNAi) technology,for the production of human-therapeutic glycoproteins

Background  

Patatins encoded by a multi-gene family are one of the major storage glycoproteins in potato tubers. Potato tubers have recently emerged as bioreactors for the production of human therapeutic glycoproteins (vaccines). Increasing the yield of recombinant proteins, targeting the produced proteins to specific cellular compartments, and diminishing expensive protein purification steps are important research goals in plant biotechnology. In the present study, potato patatins were eliminated almost completely via RNA interference (RNAi) technology to develop potato tubers as a more efficient protein expression system. The gene silencing effect of patatins in the transgenic potato plants was examined at individual isoform levels.