An easily regenerable enzyme reactor prepared from polymerized high internal phase emulsions.

A large-scale high-efficient enzyme reactor based on polymerized high internal phase emulsion monolith (polyHIPE) was prepared. First, a porous cross-linked polyHIPE monolith was prepared by in-situ thermal polymerization of a high internal phase emulsion containing styrene, divinylbenzene and polyglutaraldehyde. The enzyme of TPCK-Trypsin was then immobilized on the monolithic polyHIPE. The performance of the resultant enzyme reactor was assessed according to the conversion ability of Nα-benzoyl-l-arginine ethyl ester to Nα-benzoyl-l-arginine, and the protein digestibility of bovine serum albumin (BSA) and cytochrome (Cyt-C). The results showed that the prepared enzyme reactor exhibited high enzyme immobilization efficiency and fast and easy-control protein digestibility. BSA and Cyt-C could be digested in 10 min with sequence coverage of 59% and 78%, respectively. The peptides and residual protein could be easily rinsed out from reactor and the reactor could be regenerated easily with 4 M HCl without any structure destruction. Properties of multiple interconnected chambers with good permeability, fast digestion facility and easily reproducibility indicated that the polyHIPE enzyme reactor was a good selector potentially applied in proteomics and catalysis areas.

Development of continuous microwave-assisted protein digestion with immobilized enzyme.

In this study, an easy and efficiency protein digestion method called continuous microwave-assisted protein digestion (cMAED) with immobilized enzyme was developed and applied for proteome analysis by LC-MS(n). Continuous microwave power outputting was specially designed and applied. Trypsin and bromelain were immobilized onto magnetic micropheres. To evaluate the method of cMAED, bovine serum albumin (BSA) and protein extracted from ginkgo nuts were used as model and real protein sample to verify the digestion efficiency of cMAED. Several conditions including continuous microwave power, the ratio of immobilized trypsin/BSA were optimized according to the analysis of peptide fragments by Tricine SDS-PAGE and LC-MS(n). Subsequently, the ginkgo protein was digested with the protocols of cMAED, MAED and conventional heating enzymatic digestion (HED) respectively and the LC-MS(n) profiles of the hydrolysate was compared. Results showed that cMAED combined with immobilized enzyme was a fast and efficient digestion method for protein digestion and microwave power tentatively affected the peptide producing. The cMAED method will be expanded for large-scale preparation of bioactive peptides and peptide analysis in biological and clinical research.

Neuroprotective effects of lentivirus-mediated aquaporin-4 gene silencing in rat model of traumatic brain injury.

BACKGROUND: Traumatic brain injury (TBI) is a common clinical condition caused by external force. Aquaporin-4 (AQP4) in astrocytes participates in the generation of cell swelling in TBI. METHODS: This research explored the effect of AQP4 gene silencing in a TBI rat model. A hydraulic craniocerebral trauma instrument was employed for establishing the TBI rat model. AQP4 expression in the brain was inhibited by the injection of AQP4 shRNA-lentiviral vector. The expression of relative genes was evaluated by Western blot and qRT-PCR. Neuronal apoptosis was analyzed by TUNEL assay. RESULTS: AQP4 shRNA treatment inhibited AQP4 expression in the brain of rats with TBI. AQP4 shRNA alleviated TBI-induced brain edema and neurological deficit in rats. Neuronal apoptosis and astrocyte activation in TBI rats were reduced by AQP4 silencing. CONCLUSION: This research demonstrated that AQP4 shRNA-induced silencing of AQP4 in the TBI rat model reduced the expression of AQP4 and GFAP, alleviated brain edema, neurological deficit, neuronal apoptosis and inhibited astrocyte activation.

Novel regenerative large-volume immobilized enzyme reactor: preparation, characterization and application.

A novel large-volume immobilized enzyme reactor (IMER) on small column was prepared with organic-inorganic hybrid silica particles and applied for fast (10 min) and oriented digestion of protein. At first, a thin enzyme support layer was formed in the bottom of the small column by polymerization with α-methacrylic acid and dimethacrylate. After that, amino SiO2 particles was prepared by the sol-gel method with tetraethoxysilane and 3-aminopropyltriethoxysilane. Subsequently, the amino SiO2 particles were activated by glutaraldehyde for covalent immobilization of trypsin. Digestive capability of large-volume IMER for proteins was investigated by using bovine serum albumin (BSA), cytochrome c (Cyt-c) as model proteins. Results showed that although the sequence coverage of the BSA (20%) and Cyt-c (19%) was low, the large-volume IMER could produce peptides with stable specific sequence at 101-105, 156-160, 205-209, 212-218, 229-232, 257-263 and 473-451 of the amino sequence of BSA when digesting 1mg/mL BSA. Eight of common peptides were observed during each of the ten runs of large-volume IMER. Besides, the IMER could be easily regenerated by reactivating with GA and cross-linking with trypsin after breaking the -C=N- bond by 0.01 M HCl. The sequence coverage of BSA from regenerated IMER increased to 25% comparing the non-regenerated IMER (17%). 14 common peptides. accounting for 87.5% of first use of IMER, were produced both with IMER and regenerated IMER. When the IMER was applied for ginkgo albumin digestion, the sequence coverage of two main proteins of ginkgo, ginnacin and legumin, was 56% and 55%, respectively. (Reviewer 2) Above all, the fast and selective digestion property of the large-volume IMER indicated that the regenerative IMER could be tentatively used for the production of potential bioactive peptides and the study of oriented protein digestion.