Guoqing Ying

Selective carboxypropionylation of chitosan: synthesis, characterization, blood compatibility, and degradation.

Two water-soluble chitosan (WSC) derivatives of N-succinyl-chitosan (NSCS) and N,O-succinyl-chitosan (NOSCS) with a degree of substitution (DS) that ranged form 0.28 to 0.61 were selectively synthesized by varying the molar ration of succinic anhydride and chitosan. The chemical structure and physical properties of the chitosan derivatives were characterized by FT-IR, 1H NMR, and XRD. XRD analysis showed that the derivatives were amorphous. The lysozyme enzymatic degradation results revealed that the NSCS was of higher susceptibility to lysozyme. The degradation rate and the solubility of the chitosan derivatives were strongly determined by the degree of substitution and the position of the substitution. The results of antithrombotic properties, hemolytic properties and anticoagulant properties of WSCs indicated that the blood compatibility was dramatically improved, and the carboxyl group introduced on the C-6 or C-2 hydroxyl group appeared to impact anticoagulant activity in different ways.

Nuclease p1 immobilized on deae cellulose

Effects of various factors, such as pH, ionic strength, glutaraldehyde concentration, enzyme amount and immobilization time, on enzyme activity were investigated. The immobilization conditions were optimized by orthogonal experiments. Characterizations of immobilized nuclease p1 were also evaluated. Through orthogonal optimization, the optimal immobilization conditions were as follows: pH 5.6, ionic strength 0.125, glutaraldehyde concentration 0.20% and immobilization time 2.0 h. Optimal pH of immobilized enzyme was 5.8. Optimal temperature of immobilized enzyme was 70oC. Thermal, operational and storage stabilities of the enzyme were improved after it was immobilized on DEAE cellulose. Michaelis constant Km of immobilized enzyme at 69oC was found to be 27.21 g/l by the Lineweaver-Burk plot.

Purification and characterization of β-agarase from Paenibacillus sp.

Abstractβ-Agarase produced by Paenibacillus sp. WL (agarase WL) was purified using a combination of ammonium sulfate precipitation, DEAE-ion exchange, and gel-filtration chromatography. The purity of the agarase was increased by 11.9× with a recovery of 5.1% and a specific activity of 4,670.1 U/mg of protein. The molecular mass of the purified agarase was approximately 30 kDa (SDS-PAGE). The agarase was stable at temperature below 50°C and the favorable agar-hydrolysis activity was at 40°C. The agarase was active in the range of pH 5.0 to 8.0, and the optimal agar-hydrolysis pH value was approximately 6.0. Metal ions normally found in seawater (Na+, K+, Ca2+, Mg2+, and Al3+) could activate agarase WL. The Michaelis-Menten constant Km and maximal reaction velocity Vmax of purified agarase WL were 3.22 mg/mL and 41.5 μg/mL·min, respectively. The agarase WL was highly agar specific.

Separation and quantification of neoagaro-oligosaccharides

Oligosaccharides were obtained from agar by enzymatic hydrolysis. Activated carbon adsorption separation was used to extract oligosaccharides, and gel chromatography separation was applied to further purify oligosaccharides. The result showed that activated carbon adsorption could remove the most salt impurities, and gel column chromatography could give the separation of the two kinds of oligosaccharides. ESI-MS, 13C-NMR revealed that the molecular weight (Mw) of two oligosaccharides were 630 and 936, which were identified as neoagarotetraose and neoagarohexaose respectively.

IgG PURIFICATION USING AFFINITY FILTRATION WITH SULFAMETHAZINE-AFFINITY CARRIERS

Immunoglobulin G (IgG) antibodies are used extensively for analytical, diagnostic, and therapeutic applications. However, there are some disadvantages to purify IgG antibodies by protein A and G affinity chromatography. Therefore, it is necessary to find an effective alternative and nonchromatographic method to purify IgG. Dextran microparticles were activated and coupled with sulfamethazine to form sulfamethazine-affinity carriers. Then the carriers were used to purify IgG by affinity filtration. Quantitative and qualitative determination proved that sulfamethazine would successfully bond to the surface of dextran microparticles with a density of 85.5 μmol/g (wet). Affinity carriers were proved to withstand high shear force and reveal rare sulfamethazine leakage under filtration conditions between pH 3 to 11. The maximum IgG-binding capacity of affinity carriers was 8.03 mg IgG/g (wet). The affinity filtration process obtained a recovery yield above 80% and purity above 90%. Thus, this work involved in both the advantages of membrane filtration and affinity purification. The results, for the first time, proved that it is possible to use the small ligand sulfamethazine for affinity filtration of IgG. It is an attractive alternative to conventional protein A or G affinity chromatography.

Preparation of recombinant human thymic stromal lymphopoietin protein

ABSTRACT Human thymic stromal lymphopoietin (hTSLP) protein plays a central role in inflammation. Characterizing properties of hTSLP requires a recombinant overexpression system that produces correctly folded, active hTSLP. In this report, an efficient overexpression system for the production of hTSLP was developed. We constructed expression plasmids of the full-length hTslp gene with or without the signal peptide and transformed the plasmids into Escherichia coli. The design of the recombinant proteins included an N-terminal His-tag, which facilitated purification. An affinity gradient elution method was used to improve recovery and concentration levels of denatured hTSLP, with 90% purity observed following affinity chromatography. Refolding of the denatured hTSLP was tested using four different protein refolding approaches. The optimal refolding conditions involved stepwise buffer exchanges to reduce the urea concentration from 4 to 0 M in 50 mM Tris (pH 8.0), 1 mM EDTA, 50 mM NaCl, 10% glycerol, 400 mM L-Arg, 0.2 mM oxidized glutathione, and 2 mM reduced glutathione. The activity of the refolded recombinant hTSLP protein was measured by an ELISA assay. Interestingly, the presence of N-terminal signal peptide inhibited the overexpression of hTSLP in E. coli. The amount of recombinant hTSLP protein purified reached a level of 2.52 × 10−3 mg/L.

Preparation and Characterization of Magnetic Chitosan Microspheres for Endotoxin Adsorption

A novel magnetic chitosan microsphere (MCM) was prepared from chitosan and Fe3O4 through inverse suspension crosslinking, epichlorohydrin activation, and grafted with ligands. The magnetometer measurements data indicated that the magnetic microspheres had superparamagnetic property as well as fast magnetic response without the magnetic hysteresis effect and thus can be well used in magnetic separation. The adsorption results showed that the magnetic chitosan microsphere grafted with ligand had good adsorption capacity on endotoxin up to 554.1 EU/g under the condition of 25.0 mg/mL adsorbent, 17.2 EU/mL endotoxin. It is expected that this new microsphere will have a feasible and useful application in adsorbing endotoxin from biological products.