Soluble Prokaryotic Overexpression and Purification of Human GM-CSF Using the Protein Disulfide Isomerase b′a′ Domain

Granulocyte-macrophage colony-stimulating factor (GM-CSF) is a member of the colony-stimulating factor (CSF) family, which functions to enhance the proliferation and differentiation of hematopoietic stem cells and other hematopoietic lineages such as neutrophils, dendritic cells, or macrophages. These proteins have thus generated considerable interest in clinical therapy research. A current obstacle to the prokaryotic production of human GM-CSF (hGM-CSF) is its low solubility when overexpressed and subsequent complex refolding processes. In our present study, the solubility of hGM-CSF was examined when combined with three N-terminal fusion tags in five E. coli strains at three different expression temperatures. In the five E. coli strains BL21 (DE3), ClearColi BL21 (DE3), LOBSTR, SHuffle T7 and Origami2 (DE3), the hexahistidine-tagged hGM-CSF showed the best expression but was insoluble in all cases at each examined temperature. Tagging with the maltose-binding protein (MBP) and the b′a′ domain of protein disulfide isomerase (PDIb′a′) greatly improved the soluble overexpression of hGM-CSF at 30 °C and 18 °C. The solubility was not improved using the Origami2 (DE3) and SHuffle T7 strains that have been engineered for disulfide bond formation. Two conventional chromatographic steps were used to purify hGM-CSF from the overexpressed PDIb′a′-hGM-CSF produced in ClearColi BL21 (DE3). In the experiment, 0.65 mg of hGM-CSF was isolated from a 0.5 L flask culture of these E. coli and showed a 98% purity by SDS-PAGE analysis and silver staining. The bioactivity of this purified hGM-CSF was measured at an EC₅₀ of 16.4 ± 2 pM by a CCK8 assay in TF-1 human erythroleukemia cells.     

Fabrication of Biodegradable Polyurethane Foam Scaffolds with Customized Shapes and Controlled Mechanical Properties by Gas Foaming Technique

Poly(ε-caprolactone) (PCL)-based polyurethane (PU) foam scaffolds with different mechanical properties are fabricated using a gas foaming technique to use as porous substitutes for ear or bone with cartilage. PCL diol or triol is used as a polyol in PU foam for biocompatibility and biodegradation, with an aqueous gelatin solution as a blowing agent. The highly porous inner and outer structures of the scaffolds are developed by employing a silicone surfactant and sulfuric acid, respectively. The PU scaffolds prepared by PCL diol show ductile and flexible properties, whereas the PU scaffolds prepared by PCL triol exhibit high compression strength. In vitro test reveals the low toxicity of the PU scaffolds and the high ALP activity of MC3T3-E1 cells in the PU scaffold prepared by PCL triol. By taking advantage of the difference in mechanical properties, customized PU scaffolds with ear or bone shapes are fabricated using a silicone mold. The PU scaffolds with two compartments of PCL diol and triol (corresponding to cartilage and bone, respectively) are fabricated as a substitute for bone with cartilage. It is believed that the PU scaffolds with highly porous structure and controlled mechanical properties have wide potential application for tissue engineering.     

Blends of chlorinated isotactic polypropylene with poly(ethylene-co-vinyl acetate) II. Phase studies of the miscible blends

By measuring T_g, T_m and T_c (cloud point) phase diagrams for the four miscible blends of chlorinated isotactic polypropylenes (chlorine content 39.2 (CPP-40) and 49.8 wt.-% (CP-50)) with Poly(ethylene-co-vinyl acetate)s (vinyl acetate contents 40 (EVA-40) and 45 wt.-% (EVA-45)) were investigated. The blend of CPP-50 with EVA-40 was the most compatible of the four blend pairs.     

Preparation of ketoprofen‐loaded high‐molecular‐weight poly(vinyl alcohol) gels

High‐molecular‐weight atactic poly(vinyl alcohol) (a‐PVA) gels loaded with (R,S)‐2‐(3‐benzoylphenyl)propionic acid (ketoprofen) were prepared from 5, 6, 7, and 8 g/dL solutions of a‐PVA with a number‐average degree of polymerization of 4000 in an ethylene glycol/water mixture with an aging method to identify the effect of the initial polymer concentration on the swelling behavior, morphology, and thermal properties of a‐PVA gels. Then, the release behavior of ketoprofen from a‐PVA gels was investigated. As the polymer concentration decreased, the ability for network formation decreased, and the degree of swelling of the a‐PVA gels increased. In addition, the enthalpy increased with an increase in the a‐PVA concentration, but the melting temperatures of the gels prepared at different initial polymer concentrations were the same; this indicated that tighter gel networks would be formed by a higher polymer chain density. © 2007 Wiley Periodicals, Inc. J Appl Polym Sci 2007     

Ozonolysis of cyclododecene

Ozonolysis of cyclododecene was carried out to produce an w-formyl carboxylic acid (12-oxododecanoic acid) which is derived from zwitterion and aldehyde moiety that are formed during the reaction. The ozonolysis was performed to examine the product distribution under such reaction variables as temperature, kinds of solvent, and presence of catalyst. The yield of polymeric ozonide, which is undesirable product, was measured to be dominantly 86% without pyridine catalyst, whereas, only 10.25% with the catalyst. The optimum reaction condition was to be in MC (methylene chloride) solvent, and in the presence of equimolar olefin and pyridine catalyst at O°C, at which the yields of polymeric ozonide, 1,12-dodecanedialdehyde, 1,12-dodecanedicarboxylic acid, and 12-oxo-dodecanoic acid were 10.25%, 26.72%, 26.31%, and 36.72%, respectively.     

Viscosity effect in polyolefin ternary blends and composites

Ternary blends of PP (80) /rubber (EPM, EPDM) (10) / PE (10) and PP (80) / rubber (10) / CaCO₃ (10) composites were prepared in a twin-screw extruder. With polyethylene (PE) viscosity comparable to, or higher than that of rubber, the dispersed phase formed a reticulate structure with reduced size. On the contrary, when the viscosity of PE was significantly lower than that of rubber, the dispersed phase formed almost homogeneous morphology. With reticulate morphology, PE crystallinity content, hardness, modulus, and elongation at break of the ternary blend increased. In polypropylene (PP) / rubber / CaCO₃ composites, better dispersion of CaCO₃ in the PP matrix was obtained when the viscosity of rubber was significantly higher than that of matrix. With better dispersion, hardness and tensile properties were improved, but the impact strength more or less decreased. © 1993 John Wiley & Sons, Inc.     

Biodegradability and mechanical properties of agro‐flour–filled polybutylene succinate biocomposites

The objective of this study was the production of rice husk flour (RHF) and wood flour (WF) filled polybutylene succinate (PBS) biocomposites as alternatives to cellulosic material filled conventional plastic (polyolefins) composites. PBS is one of the biodegradable polymers, made from the condensation reaction of 1,4‐butanediol and succinic acid that can be naturally degraded in the natural environment. We compared the mechanical properties between conventional plastics and agro‐flour–filled PBS biocomposites. We evaluated the biodegradability and mechanical properties of agro‐flour–filled PBS biocomposites according to the content and filler particle size of agro‐flour. As the agro‐flour loading was increased, the tensile and impact strength of the biocomposites decreased. As the filler particle size decreased, the tensile strength of the biocomposites increased but the impact strength decreased. The addition of agro‐flour to PBS produced a more rapid decrease in the tensile strength, notched Izod impact strength, and percentage weight loss of the biocomposites during the natural soil burial test. These results support the application of biocomposites as environmentally friendly materials. © 2005 Wiley Periodicals, Inc. J Appl Polym Sci 97: 1513–1521, 2005     

Resistivity and thermal reproducibility of carbon black and metallic powder filled silicone rubber heaters

Flexible heaters were prepared by extruding platinum‐catalyzed silicone rubber composites with conductive carbon black (CB) and metallic fillers. The conductor resistivity of the extruded heaters decreased in order of conductive titanium dioxide (TiO₂) > aluminum powder ≈ zinc powder > copper powder. Thermoelectric switching phenomena were investigated for the silicone rubber/CB/metallic powder systems. The positive temperature coefficient effect was dependent mainly on the CB content rather than on the content of the metallic powders. Resistivity and thermal reproducibility of the extruded heaters were also investigated by periodically applying AC voltage of 110 V. The heaters containing copper and TiO₂ powders exhibited excellent electrical reproducibility. © 2005 Wiley Periodicals, Inc. J Appl Polym Sci 95: 1122–1128, 2005     

Low‐temperature photoinitiation solution polymerization behavior of N‐vinylcarbazole in tetrahydrofuran

N‐Vinylcarbazole (VCZ) was solution‐polymerized in tetrahydrofuran (THF) at ?20, 0, and 20°C using the photoinitiation method; the effects of the amount of solvent, polymerization temperature, and photoinitiator concentration were investigated. On the whole, the experimental results corresponded to predicted ones. Low polymerization temperature using photoinitiation proved to be successful in obtaining poly(N‐vinylcarbazole) (PVCZ) of a high molecular weight with a smaller temperature rise during polymerization; nevertheless of free radical polymerization by 2,2′‐azobis(2,4‐dimethylvaleronitrile) (ADMVN). The photo‐solution polymerization rate of VCZ in THF was proportional to the 0.47 power of ADMVN concentration. The molecular weight was higher and the molecular weight distribution was narrower with PVCZ polymerized at lower temperatures. For PVCZ prepared in THF at ?20°C using a photoinitiator concentration of 0.00005 mol/mol of VCZ, a weight‐average molecular weight of 510,000 was obtained, with a polydispersity index of 1.73, and a degree of lightness converged to about 99%. © 2002 Wiley Periodicals, Inc. J Appl Polym Sci 86: 3667–3672, 2002