Cell disruption enhanced the pure EGFP recovery from an EGFP-intein-surface protein production system in recombinant E. coli

In this study, three approaches for protein production were devised to enhance the efficiencies in yielding high purity protein via surface display system. A plasmid carrying enhanced green fluorescent protein (EGFP), an intein (INT) and ice nucleation protein (INP) was constructed to produce EGFP via surface display in Escherichia coli. To obtain high purity of the produced EGFP, several procedures, including osmotic shock, surfactant addition and cell disruption were employed. Among these approaches, the cell disruption method gave the highest EGFP purity by simply conducting several centrifugations. An EGFP yield of 63 mg/L with 97% purity was obtained. The result demonstrated that pure EGFP can be harvested only through centrifugation; no complicated processes or expensive equipment are required. This approach shows potential for the production of pure recombinant proteins in scale-up processes for biotechnological, academic and industrial uses

Stomach-Specific Controlled Release Gellan Beads of Acid-Soluble Drug Prepared by Ionotropic Gelation Method

The purpose of the present work was the development and evaluation of stomach-specific controlled release mucoadhesive drug delivery system prepared by ionotropic gelation of gellan beads, containing acid-soluble drug amoxicillin trihydrate, using 32 factorial design with concentration of gellan gum and quantity of drug as variables. The study showed that beads prepared in alkaline cross-linking medium have higher entrapment efficiency than the acidic cross-linking medium. The entrapment efficiency was in the range of 32% to 46% w/w in acidic medium, which increased up to 60% to 90% w/w in alkaline medium. Batches with lowest, medium, and highest drug entrapment were subjected to chitosan coating to form a polyelectrolyte complex film. As polymer concentration increases, entrapment efficiency and particle size increases. Scanning electron microscopy revealed spherical but rough surface due to leaching of drug in acidic cross-linking solution, dense spherical structure in alkaline cross-linking solution, and rough surface of chitosan-coated beads with minor wrinkles. The in vitro drug release up to 7 h in a controlled manner following the Peppas model (r = 0.9998). In vitro and in vivo mucoadhesivity study showed that beads have good mucoadhesivity and more than 85% beads remained adhered to stomach mucosa of albino rat even after 7 h. In vitro growth inhibition study showed complete eradication of Helicobacter pylori. These results indicate that stomach-specific controlled release mucoadhesive system of amoxicillin gellan beads may be useful in H. pylori treatment

Inhibitory effect of <it>Bifidobacterium infantis</it>-mediated sKDR prokaryotic expression system on angiogenesis and growth of Lewis lung cancer in mice

<p>Abstract</p> <p>Background</p> <p>To construct the <it>Bifidobacterium infantis</it>-mediated soluble kinase insert domain receptor (sKDR) prokaryotic expression system and to observe its inhibitory effect on growth of human umbilicus vessel endothelial cells (HUVECs) <it>in vitro</it> and Lewis lung cancer (LLC) on mice <it>in vivo.</it></p> <p>Methods</p> <p>The <it>Bifidobacterium infantis</it>-mediated sKDR prokaryotic expression system was constructed through electroporation and subsequently identified through PCR and Western blot analysis. HUVECs were added to the products of this system to evaluate the anti-angiogenesis effect through MTT assay <it>in vitro</it>. The LLC mice models were divided into three groups: one group treated with saline (group a); one group treated with recombinant <it>Bifidobacterium infantis</it> containing pTRKH2-PsT plasmid group (group b); and one group treated with recombinant <it>Bifidobacterium infantis</it> containing pTRKH2-PsT/sKDR plasmid group (group c). The quality of life and survival of mice were recorded. Tumor volume, tumor weight, inhibitive rate, and necrosis rate of tumor were also evaluated. Necrosis of tumor and signals of blood flow in tumors were detected through color Doppler ultrasound. In addition, microvessel density (MVD) of the tumor tissues was assessed through CD31 immunohistochemical analysis.</p> <p>Results</p> <p>The positively transformed <it>Bifidobacterium infantis</it> with recombinant pTRKH2-PsT/sKDR plasmid was established, and was able to express sKDR at gene and protein levels. The proliferation of HUVECs cultivated with the extract of positively transformed bacteria was inhibited significantly compared with other groups (P < 0. 05). The quality of life of mice in group c was better than in group a and b. The recombinant <it>Bifidobacterium infantis</it> containing pTRKH2-PsT/sKDR plasmid enhanced the efficacy of tumor growth suppression and prolongation of survival, increased the necrosis rate of tumor significantly, and could obviously decrease MVD and the signals of blood flow in tumors.</p> <p>Conclusion</p> <p>The <it>Bifidobacterium infantis</it>-mediated sKDR prokaryotic expression system was constructed successfully. This system could express sKDR at gene and protein levels and significantly inhibit the growth of HUVECs induced by VEGF <it>in vitro</it>. Moreover, it could inhibit tumor growth and safely prolong the survival time of LLC C57BL/6 mice.</p

Redox-Sensitive Disassembly of Amphiphilic Copolymer Based Micelles

Amphiphilic polymers of different hydrophilic lipophilic ratios were prepared by free radical polymerization using two monomers consisting of triethylene glycol as the hydrophilic part and an alkyl chain connected by disulfide bond as the hydrophobic part. These polymers form micelle-like nanoassemblies in aqueous media and can encapsulate hydrophobic drug molecules up to 14% of their mass. In a reducing environment, these polymeric micelles disassemble and dissolve in water, since the amphiphilic polymers are converted into hydrophilic polymers upon cleavage of the disulfide bond. This disassembly event results in the release of hydrophobic molecules that had been encapsulated inside the micelle, the rate of which was found to be dependent on the concentration of the reducing agent, glutathione (GSH). In vitro experiments also show that the GSH-dependent release of the doxorubicin can be used to effect cytotoxicity in MCF-7 cells