Comparative studies of the artificial chaperone-assisted refolding of thermally denatured bovine carbonic anhydrase using different capturing ionic detergents and beta-cyclodextrin

Artificial chaperone-assisted refolding has been shown to be an effective approach for improving the refolding yield of some of the denatured proteins. Since identical concentrations of various detergents do not induce similar variations in the protein structures, we arranged to evaluate the artificial chaperoning capabilities of several ionic detergents as a function of charge, structure, and the hydrophobic tail length of the detergent. Our results indicate that carbonic anhydrase can be refolded from its denatured state via artificial chaperone strategy using both anionic and cationic detergents. However, the extent of refolding assistance (kinetic and refolding yield) were different due to protein and detergent net charges, detergent concentrations, and the length of hydrophobic portion of each detergent. These observed differences were attributed to physical properties of CA-detergent complexes and/or to the kinetics of detergent stripping by beta-cyclodextrin from the protein-detergent complexes which is apparently dependent on the detergent-beta-CD association constants and the nature of the partially stripped complexes.     

Purification of bacteriophage M13 by anion exchange chromatography

M13 is a non-lytic filamentous bacteriophage (phage). It has been used widely in phage display technology for displaying foreign peptides, and also for studying macromolecule structures and interactions. Traditionally, this phage has been purified by cesium chloride (CsCl) density gradient ultracentrifugation which is highly laborious and time consuming. In the present study, a simple, rapid and efficient method for the purification of M13 based on anion exchange chromatography was established. A pre-packed SepFast™ Super Q column connected to a fast protein liquid chromatography (FPLC) system was employed to capture released phages in clarified Escherichia coli fermented broth. An average yield of 74% was obtained from a packed bed mode elution using citrate buffer (pH 4), containing 1.5 M NaCl at 1 ml/min flow rate. The purification process was shortened substantially to less than 2 h from 18 h in the conventional ultracentrifugation method. SDS-PAGE revealed that the purity of particles was comparable to that of CsCl gradient density ultracentrifugation method. Plaque forming assay showed that the purified phages were still infectious.     

Desorption of the cellulase systems of Trichoderma reesei and a Botrytis sp. from Avicel

Maximum desorptions (96%) of two microbial cellulolytic enzyme systems (Trichoderma reesei and a Botrytis sp.) from the Avicel were achieved using glycerol / diethyl ether (1:1, v/v). Then, using n-butanol, the cellulolytic enzyme systems were easily recovered from the glycerol / diethyl ether mixture. © Rapid Science Ltd. 1998     

Anti-amyloidogenic effect of AA3E2 attenuates β-amyloid induced toxicity in SK-N-MC cells

β-Amyloid peptide (Aβ) is believed to play a recognized role in pathogenesis of Alzheimer's disease (AD). Self-association of Aβ peptide into amyloid fibrils causes neurotoxicity. Compounds capable of interfering with Aβ–Aβ interaction through binding to nucleation sites can inhibit Aβ amyloidogenesis and Aβ-induced cytotoxicity. AA3E2 is a triazine-derivative whose anti-amyloidogenic ability has previously been established. In the present study, we evaluated the protective effect of AA3E3 against Aβ_1–42-induced toxicity in SK-N-MC cell line. The cell exposure to the co-incubated Aβ_1–42 with AA3E2 decreased the cell viability loss dose-dependently, compared to cells exposed to Aβ_1–42 fibrils.Co-incubation with AA3E2 also attenuated the ROS production, activation of caspase-3 and the extent of apoptotic cell death induced by Aβ_1–42 fibril. Moreover, the 3D structure of the molecular associates between Aβ_1–42 and AA3E2 were theoretically determined by docking studies. Our docking data indicated that AA3E2 inhibits the formation of Aβ fibril likely via binding to the nucleation site within the hydrophobic region of Aβ (KLVFF). These observations provide the background for future design of more elegant β-breaking agents for dissolution of Aβ fibrillar aggregates.