Gelatin-immobilised poly(hydroxyethyl methacrylate) cryogel for affinity purification of fibronectin.

In this work, fibronectin purification from human plasma with the gelatin-immobilised poly(hydroxyethyl methacrylate) (PHEMA) cryogel has been evaluated. The PHEMA cryogel was prepared by cryo-polymerisation which proceeds in an aqueous solution of monomer frozen inside a plastic syringe. The PHEMA cryogel contained interconnected macrochannels of 10-200 µm in diameter. Gelatin molecules were covalently immobilised onto the PHEMA cryogel via carbodiimide activation. The gelatin-immobilised PHEMA cryogel was used to purify fibronectin from human plasma. Fibronectin adsorption from human plasma on the PHEMA cryogel was 0.30 mg/ml, while much higher adsorption values, up to 38 mg/ml, was obtained with the gelatin-immobilised PHEMA cryogel. The fibronectin adsorption capacity of the gelatin-immobilised PHEMA cryogel did not change with an increase in the flow rate of plasma. Up to 92 % of the adsorbed fibronectin was eluted using 2 M urea containing 1 M NaCl as elution agent. The adsorption-elution cycle was repeated ten times using the same PHEMA cryogel. No remarkable decrease was detected in the adsorption capacity of the gelatin-immobilised PHEMA cryogel.

Molecular and clinical evaluation of Turkish patients with lysinuric protein intolerance.

Lysinuric protein intolerance is an autosomal recessive metabolic disorder caused by defective transport of the cationic amino acids lysine, arginine and ornithine in the epithelial cells of the basolateral membrane in the small intestine and renal tubules. Mutations in the solute carrier family 7, member 7, SLC7A7, gene cause this multisystemic disease with a variety of clinical symptoms such as hepatosplenomegaly, osteoporosis, hypotonia, developmental delay, pulmonary insufficiency or end-stage renal disease. In the present study, genomic structure of SLC7A7 in six Turkish patients with lysinuric protein intolerance was examined in order to detect disease causing mutations by denaturing high pressure liquid chromatography and direct sequencing. Four novel mutations were identified in SLC7A7: c.223insGTC, p.Val74_Ile75insVal; c.283insTGG, p.Glu94_Thr95insTrp; c.344_347delTTGC, p.Leu115LeufsX53; and c.1099insT, p.Ile367TyrfsX16. Clinical and biochemical findings were evaluated together with these molecular analyses.

Purification of urease from jack bean (Canavalia ensiformis) with copper (II) chelated poly(hydroxyethyl methacrylate-N-methacryloyl-(L)-histidine methyl ester) cryogels.

Jack bean (Canavalia ensiformis) is the source of interesting proteins that contribute to modern biochemistry, and urease is the primary of these proteins. Owing to its role and occurrence in nature, urease has become a part of extensive studies. In this study, jack bean urease (JBU) was purified by immobilized metal affinity chromatography using Cu(2+) chelated poly(hydroxyethyl methacrylate-N-methacryloyl-(L)-histidine methyl ester) [PHEMAH-Cu(2+)]-based cryogels. PHEMAH-Cu(2+) cryogel was synthesized and characterized for swelling degree, morphology (by SEM), N-methacryloyl-(L)-histidine methyl ester and Cu(2+) incorporation (by elemental analysis and atomic absorption spectrophotometry). The binding of JBU to PHEMAH-Cu(2+) cryogel was optimized by examining the effect of pH, flow rate and JBU concentration on binding. The maximal binding of JBU was 23.2 mg/dry gram of adsorbent. The maximal binding of JBU extracted from jack bean meal was 67.8 mg/dry gram of adsorbent. The elution of JBU from cryogel column was accomplished by 1.0 M NaCl in 20 mM phosphate buffer (pH 8.0). Molecular weight and purity of JBU from jack bean meal was estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. It was observed that JBU could be repeatedly bound and eluted from (PHEMAH)-Cu(2+) cryogel with less than 10% loss in column capacity.

Mannose-specific lectin isolation from Canavalia ensiformis seeds by PHEMA-based cryogel.

Mannose-specific lectin Concanavalin A (Con A) was purified from Canavalia ensiformis seeds. For this purpose, mannose attached poly(hydroxyethyl methacrylate) (PHEMA) cryogel was prepared by cryopolymerization. Mannose was used as the affinity ligand and was covalently attached onto the PHEMA cryogel via carbodiimide activation. The PHEMA cryogel containing 23.3 mmol mannose/g polymer were used in the binding studies. Con A binding with the mannose attached PHEMA cryogel from Con A aqueous solution was 5.2 mg/g at pH 7. Maximum binding capacity for Con A from C. ensiformis seed extract was 39 mg/g. Con A was eluted with 0.3 M galactose, and the purity of Con A was determined by sodium dodecyl sulfate polyacrylamide gel electrophoresis. It was observed that the mannose attached PHEMA cryogel can be used without significant decrease in Con A binding capacity after six binding-elution cycles.

Poly(hydroxyethyl methacrylate) based affinity cryogel for plasmid DNA purification.

The aim of this study is to prepare supermacroporous pseudospecific cryogel which can be used for the purification of plasmid DNA (pDNA) from bacterial lysate. N-methacryloyl-(l)-histidine methyl ester (MAH) was chosen as the pseudospecific ligand and/or comonomer. Poly(hydroxyethyl methacrylate-N-methacryloyl-(l)-histidine methyl ester) [PHEMAH] cryogel was produced by free radical polymerization initiated by N,N,N',N'-tetramethylene diamine (TEMED) and ammonium persulfate (APS) pair in an ice bath. Compared with the PHEMA cryogel (50 µg/g polymer), the pDNA adsorption capacity of the PHEMAH cryogel (13,350 µg/g polymer) was improved significantly due to the MAH incorporation into the polymeric matrix. The amount of pDNA bound onto the PHEMAH cryogel disks first increased and then reached a saturation value (i.e., 13,350µg/g) at around 300 µg/ml pDNA concentration. pDNA adsorption amount decreased from 1137 µg/g to 160 µg/g with the increasing NaCl concentration. The maximum pDNA adsorption was achieved at 25 °C. The overall recovery of pDNA was calculated as 90%. The PHEMAH cryogel could be used 3 times without decreasing the pDNA adsorption capacity significantly. The results indicate that the PHEMAH cryogel disks promise high selectivity for pDNA.

Controlled release of EGF and bFGF from dextran hydrogels in vitro and in vivo.

In the present study, dextran-epichlorohydrin hydrogels were employed as carriers for the controlled release of epidermal growth factor (EGF) and basic fibroblast growth factor (bFGF). The hydrogels were synthesized from 50% (by weight) monomeric cross-linker, epichlorohydrin, containing dextran mixtures by intermolecular side-chain reaction of dextran-hydroxyl groups with epichlorohydrin-epoxy groups. The hydrogel disks of 3-mm diameter and 1.5-mm thickness have a high swelling capacity (EWC = 650%) and enough mechanical stability for the studies in vivo. Impregnation of EGF and bFGF into the dried hydrogels was carried out by use of phosphate buffered saline solution (PBS, pH = 7.4) containing 0.5 microg mL(-1) EGF and 0.1 microg mL(-1) bFGF, respectively. The in vitro release of growth factors was detected by fluorescence spectroscopy. The prolonged release of EGF is continued up to the 14th day, in comparison with a 26-day release of bFGF. The in vivo studies were realized with subcutaneously implanted hydrogels in Wistar albino rats. The rate of neovascularization was analyzed statistically using one-way analysis of significance with EGF and bFGF incorporated hydrogels. In conclusion, dextran-epichlorohydrin hydrogels were shown to be an alternative delivery system for the release of growth factors.