The suppression of the glutelin storage protein gene in transgenic rice seeds results in a higher yield of recombinant protein

Glutelin is a major seed storage protein, accounting for 60?C80?% of the total endosperm protein content in rice. To test whether we could augment the expression of an introduced recombinant protein in rice by suppressing the glutelin gene, we generated transgenic glutelin RNAi (glu RNAi) rice seeds. RNA gel blot analyses confirmed that the endogenous glutelin gene was severely suppressed in these transgenic rice lines. RT-PCR analysis further revealed that all the members of glutelin multigene family were downregulated. Transgenic glu RNAi rice seeds expressing a recombinant red fluorescent protein (RFP) showed stronger fluorescence than seeds transformed with the RFP gene only. Western blot analysis further revealed that the relative accumulation of RFP in glu RNAi seeds was twofold higher than that in the RFP-only transgenic seeds. These results suggest that RNAi targeting of an endogenous storage protein could be of great utility in obtaining higher transgene expression in genetically engineered rice and other plant lines.     

Establishment and maintenance of human embryonic stem cell lines on human feeder cells derived from uterine endometrium under serum-free condition

Human embryonic stem (hES) cells are usually established and maintained on mouse embryonic fibroblast (MEFs) feeder layers. However, it is desirable to develop human feeder cells because animal feeder cells are associated with risks such as viral infection and/or pathogen transmission. In this study, we attempted to establish new hES cell lines using human uterine endometrial cells (hUECs) to prevent the risks associated with animal feeder cells and for their eventual application in cell-replacement therapy. Inner cell masses (ICMs) of cultured blastocysts were isolated by immunosurgery and then cultured on mitotically inactivated hUEC feeder layers. Cultured ICMs formed colonies by continuous proliferation and were allowed to proliferate continuously for 40, 50, and 55 passages. The established hES cell lines (Miz-hES-14, -15, and -9, respectively) exhibited typical hES cells characteristics, including continuous growth, expression of specific markers, normal karyotypes, and differentiation capacity. The hUEC feeders have the advantage that they can be used for many passages, whereas MEF feeder cells can only be used as feeder cells for a limited number of passages. The hUECs are available to establish and maintain hES cells, and the high expression of embryotrophic factors and extracellular matrices by hUECs may be important to the efficient growth of hES cells. Clinical applications require the establishment and expansion of hES cells under stable xeno-free culture systems.     

Characterization of ELP-fused ω-Transaminase and Its Application for the Biosynthesis of β-Amino Acid

Optically pure amines, β-amino acids and γ-amino acids are the valuable precursors to produce biologically active compounds. The ω-TAs are the class of enzymes which are widely used to produce such compounds. In this work (S)-ω-transaminase from the thermophilic eubacterium Sphaerobacter thermophilus (St-TA) was fused with Elastin-like polypeptides (ELPs) through the cloning process and expressed in E. coli cells. The characterization of this fusion complex was performed with respect to thermostability and effect of DMSO. Where in case of St-TA-ELP-V₆₀, major difference in the transition temperature (T_t) was observed, wherein a T_t of 38 and 70°C was observed at the increasing concentration of DMSO from 5 to 25% (v/v). Interestingly, these fusion proteins the activity was preserved even after the aggregation of fusion complex at Tt. The substrate specificity and product inhibition analysis showed that ω-TA-ELPs had comparable results as that of wild type ω-TA. Moreover, the fused ω-TA could be efficiently reused for up to 20 batches of transamination reaction. Furthermore, the applicability of the fusion protein for the production of a sitagliptin precursor (R)-3-amino-4-(2,4,5-triflurophenyl) butanoic acid (3-ATfBA) was evaluated, wherein 3-ATfBA was synthesized with good conversion (65%).     

Simulation of sequential batch reactor (SBR) operation for simultaneous removal of nitrogen and phosphorus

Modeling of the operation of sequential batch reactor (SBR) was performed to find out optimum design parameters for simultaneous removal of nitrogen and phosphorus in a small-scale wastewater treatment plant. The models were set up with material balances on SBR operation and Monod kinetics. The model parameters were obtained to best fit the experimental results in a small scale SBR. The models were useful in optimizing hydraulic retention time (HRT) and successfully simulated operations of SBR in a larger scale. Especially the model predicted well the reactions occurring in the filling period as well as the effect of dilution, and evaluated the performance of SBR process under diverse operating conditions.     

TAT-mediated delivery of human glutamate dehydrogenase into PC12 cells

Human glutamate dehydrogenase (GDH) gene was fused with a gene fragment encoding the nine amino acid (RKKRRQRRR) protein transduction domain of human immunodeficiency virus TAT protein in bacterial expression vector to produce genetic in-frame TAT-GDH fusion protein. The TAT-GDH protein can enter PC12 cells efficiently when added exogenously in culture media as determined by Western blot analysis and enzyme activities. Once inside the cells, the transduced denatured TAT-GDH protein showed a full activity of GDH indicating that the TAT-GDH fusion protein was correctly refolded after delivery into cells and the activities of GDH in the TAT-GDH fusion protein was not affected by the addition of the TAT sequence. TAT-GDH fusion protein and TAT itself showed no cytotoxicity in PC12 cells. Although the exact mechanism of transduction across a membrane remains unclear, the transduction activity of TAT-GDH into PC12 cells may suggest new possibilities for direct delivery of GDH into the patients with the GDH-deficient disorders.     

High production of D-tagatose, a potential sugar substitute, using immobilized L-arabinose isomerase

An L-arabinose isomerase of Escherichia coli was immobilized using covalent binding to agarose to produce D-tagatose, a bulking sweetener that can be economically used as a sugar substitute. The immobilized L-arabinose isomerase stably produced an average of 7.5 g-tagatose/L.day for 7 days with a productivity exceeding that of the free enzyme (0.47 vs 0.30 mg/U.day). Using a scaled-up immobilized enzyme system, 99.9 g-tagatose/L was produced from galactose with 20% equilibrium in 48 h. The process was repeated two more times with production of 104.1 and 103.5 g-tagatose/L. D-Tagatose production using an immobilized L-arabinose isomerase has a high potential for commercial application.     

Inhibition of Endoplasmic Reticulum Stress-induced Apoptosis by Silkworm Storage Protein 1

The endoplasmic reticulum (ER) plays essential roles indispensable for cellular activity and survival, including functions such as protein synthesis, secretory and membrane protein folding, and Ca²⁺ release in cells. The ER is sensitive to stresses that can lead to the aggregation and accumulation of misfolded proteins, which eventually triggers cellular dysfunction; severe or prolonged ER stress eventually induces apoptosis. ER stress-induced apoptosis causes several devastating diseases such as atherosclerosis, neurodegenerative diseases, and diabetes. In addition, the production of biopharmaceuticals such as monoclonal antibodies requires the maintenance of normal ER functions to achieve and maintain the production of high-quality products in good quantities. Therefore, it is necessary to develop methods to efficiently relieve ER stress and protect cells from ER stress-induced apoptosis. The silkworm storage protein 1 (SP1) has anti-apoptotic activities that inhibit the intrinsic mitochondrial apoptotic pathway. However, the role of SP1 in controlling ER stress and ER stress-induced apoptosis has not been investigated. In this paper, we demonstrate that SP1 can inhibit apoptosis induced by a well-known ER stress inducer, thapsigargin, by alleviating the decrease in cell viability and mitochondrial membrane potential. Interestingly, SP1 significantly blocked increases in CHOP and GRP78 expression as well as ER Ca2+ leakage into the cytosol following ER stress induction. This indicates that SP1 protects cells from ER stressinduced apoptosis by functioning as an upstream inhibitor of apoptosis. Therefore, studying SP1 function can offer new insights into protecting cells against ER stress-induced apoptosis for future applications in the biopharmaceutical and medicine industries.     

Removing environmental organic pollutants with bioremediation and phytoremediation

Hazardous organic pollutants represent a threat to human, animal, and environmental health. If left unmanaged, these pollutants could cause concern. Many researchers have stepped up efforts to find more sustainable and cost-effective alternatives to using hazardous chemicals and treatments to remove existing harmful pollutants. Environmental biotechnology, such as bioremediation and phytoremediation, is a promising field that utilizes natural resources including microbes and plants to eliminate toxic organic contaminants. This technology offers an attractive alternative to other conventional remediation processes because of its relatively low cost and environmentally-friendly method. This review discusses current biological technologies for the removal of organic contaminants, including chlorinated hydrocarbons, focusing on their limitation and recent efforts to correct the drawbacks.     

Constitutive production of human leptin by fed-batch culture of recombinant rpoS- Escherichia coli

High-level production of human leptin by fed-batch culture of recombinant Escherichia coli using constitutive promoter system was investigated. For the constitutive expression of the obese gene encoding human leptin, the strong constitutive HCE promoter cloned from the D-amino acid aminotransferase gene of Geobacillus toebii was used. To develop an optimal host-vector system, several different recombinant E. coli strains were compared for leptin production. In flask cultures, E. coli FMJ123, which is a rpoS mutant strain, showed the highest level of leptin production (41% of total proteins). By comparing the expression levels of leptin in several different rpoS- and rpoS+ strains, it could be concluded that rpoS mutation positively affected constitutive production of leptin. For the large-scale production of human leptin, fed-batch cultures of recombinant E. coli FMJ123 were carried out using three different feeding solutions--chemically defined, yeast extract-containing, and casamino acid-containing feeding solutions. Among these, the use of casamino acid-containing feeding solution allowed production of leptin up to 2.1 g/L, which was 2.1- and 1.8-fold higher than that obtained with chemically defined and yeast extract-contained feeding solutions, respectively. These results suggest that the HCE promoter can be used for the efficient production of leptin, and most likely other recombinant proteins, in a constitutive manner.     

Crystalline structure analysis of cellulose treated with sodium hydroxide and carbon dioxide by means of X-ray diffraction and FTIR spectroscopy

Crystalline structures of cellulose (named as Cell 1), NaOH-treated cellulose (Cell 2), and subsequent CO2-treated cellulose (Cell 2-C) were analyzed by wide-angle X-ray diffraction and FTIR spectroscopy. Transformation from cellulose I to cellulose II was observed by X-ray diffraction for Cell 2 treated with 15-20 wt% NaOH. Subsequent treatment with CO2 also transformed the Cell 2-C treated with 5-10 wt% NaOH. Many of the FTIR bands including 2901, 1431, 1282, 1236, 1202, 1165, 1032, and 897 cm(-1) were shifted to higher wave number (by 2-13 cm(-1)). However, the bands at 3352, 1373, and 983 cm(-1) were shifted to lower wave number (by 3-95 cm(-1)). In contrast to the bands at 1337, 1114, and 1058 cm(-1), the absorbances measured at 1263, 993, 897, and 668 cm(-1) were increased. The FTIR spectra of hydrogen-bonded OH stretching vibrations at around 3352 cm(-1) were resolved into three bands for cellulose I and four bands for cellulose II, assuming that all the vibration modes follow Gaussian distribution. The bands of 1 (3518 cm(-1)), 2 (3349 cm(-1)), and 3 (3195 cm(-1)) were related to the sum of valence vibration of an H-bonded OH group and an intramolecular hydrogen bond of 2-OH ...O-6, intramolecular hydrogen bond of 3-OH...O-5 and the intermolecular hydrogen bond of 6-O...HO-3', respectively. Compared with the bands of cellulose I, a new band of 4 (3115 cm(-1)) related to intermolecular hydrogen bond of 2-OH...O-2' and/or intermolecular hydrogen bond of 6-OH...O-2' in cellulose II appeared. The crystallinity index (CI) was obtained by X-ray diffraction [CI(XD)] and FTIR spectroscopy [CI(IR)]. Including absorbance ratios such as A1431,1419/A897,894 and A1263/A1202,1200, the CI(IR) was evaluated by the absorbance ratios using all the characteristic absorbances of cellulose. The CI(XD) was calculated by the method of Jayme and Knolle. In addition, X-ray diffraction curves, with and without amorphous halo correction, were resolved into portions of cellulose I and cellulose II lattice. From the ratio of the peak area, that is, peak area of cellulose I (or cellulose II)/total peak area, CI(XD) were divided into CI(XD-CI) for cellulose I and CI(XD-CII) for cellulose II. The correlation between CI(XD-CI) (or CI(XD-CII)) and CI(IR) was evaluated, and the bands at 2901 (2802), 1373 (1376), 897 (894), 1263, 668 cm(-1) were good for the internal standard (or denominator) of CI(IR), which increased the correlation coefficient. Both fraction of the absorbances showing peak shift were assigned as the alternate components of CI(IR). The crystallite size was decreased to constant value for Cell 2 treated at >or= 15 wt% NaOH. The crystallite size of Cell 2-C (cellulose II) was smaller than that of Cell 2 (cellulose I) treated at 5-10 wt% NaOH. But the crystallite size of Cell 2-C (cellulose II) was larger than that of Cell 2 (cellulose II) treated at 15-20 wt% NaOH.