Molecularly imprinted poly beta-cyclodextrin polymer: application in protein refolding

Regarding our previous report on refolding of alkaline phosphatase [Yazdanparast and Khodagholi, 2005 Arch. Biochem. Biophys] it was found that in spite of the anti-aggregatory effect of 3-[(3-cholamidopropyl) dimethylammonio]-1-propanesulfonate (CHAPS), a zwitteronic detergent, the recovered activity was almost the same as the recovered activity obtained through the unassisted approach. The low recovery yield is probably due to the bulky groups of the detergent that interfere with its entrance into the small cavity of the stripping agent, cyclodextrin, implying that the stripping of detergent molecules from the detergent-protein complexes plays a major role in successful refolding processes. To improve the efficiency of CHAPS stripping, we evaluated, for the first time, the stripping potential of a molecular imprinting polymer designed to replace beta-CD. In this approach, CHAPS was used as the template and the refolding of GuHCl denatured alkaline phosphatase was studied. Our results indicated that under the optimally developed refolding environment and similar to stripping by soluble beta-CD, a refolding yield of 79% was obtained for denatured alkaline phosphatase using 20 mg/ml of the molecularly imprinted poly (beta-CD) polymer. The major advantage of the new stripping agent, besides of its recycling option and ease of separation from the finished product, is its high potential of preventing aggregate formation. Based on these results, it seems that the new stripping strategy can constitute an ideal approach for refolding of proteins at much lower industrial costs compared to stripping with soluble beta-cyclodextrin.     

Evaluation of artificial chaperoning behavior of an insoluble cyclodextrin-rich copolymer: solid-phase assisted refolding of carbonic anhydrase

Insoluble beta-cyclodextrin (beta-CD) copolymers have been used for the refolding of thermally and/or chemically denatured carbonic anhydrase with refolding yield of 40% using 300 mg of the copolymer/ml refolding solution containing 0.042 mg/ml protein. In an attempt to enhance the refolding yield with lower quantities of the copolymer, a new beta-CD-rich copolymer with higher beta-CD content was synthesized. Regarding the need for rapid stripping of the detergent molecules from the detergent-protein complexes formed in the capture step of the technique (artificial chaperone-assisted refolding), experimental variables (e.g. copolymer and the protein contents) were optimized to improve the refolding yields along with depressing the aggregate formation. In addition, comparative studies using different ionic detergents and the copolymer were conducted to get a more comprehensive understanding of the detergent's tail length in the stripping step of the process. Our results indicated that under the optimal developed refolding environment, the denatured CA was refolded with a yield of 75% using only 5mg of the copolymer/1.2 ml refolding solution containing 0.0286 mg/ml protein. Taking into account the recycling potential of the copolymer, the new resin, with significant cost-cutting capability, is a suitable candidate for industrial applications.     

Control of aggregation in protein refolding: cooperative effects of artificial chaperone and cold temperature

Refolding of GuHCl-denatured recombinant-human growth hormone (r-hGH) was investigated in both dilution additive and artificial chaperone assisted modes. In both techniques, it was found that CTAB is a better additive (in dilution mode) or a capturing agent (in artificial chaperone method). Neither of the two techniques was capable of complete inhibition of aggregates formed during refolding process. In dilution, using CTAB or alpha-cyclodextrin (alpha-CD) as two different additives, the aggregation was inhibited by almost 55%. However, the extent of inhibition raised to almost 82% in artificial chaperone assisted mode using CTAB as the capturing and alpha-CD as the stripping agents. Maximum inhibition of aggregation (up to 97%) was obtained when the entire process of refolding was done at 4 degrees C. However, under this temperature program, the far-UV CD and intrinsic fluorescence spectra of the refolded samples were not superimposable on their respective native spectra. The spectra superimposibilities were obtained when the refolding process was achieved under a well worked out temperature program: incubation of the sample for 3 min at 4 degrees C after initiation of the stripping step followed by overnight incubation at 22 degrees C. Based on these data, it is expected that higher activity recovery yields of recombinant proteins, particularly at relatively higher protein concentrations, could be achieved by getting a better molecular understanding of major factors responsive for aggregation and refolding pathways.     

The glycophosphatidylinositol anchor oppositely affects unfolding and refolding of alkaline phosphatase

Regarding the world wide success of artificial chaperone-assisted protein refolding technique and based on its well worked-out mechanism, it is anticipated that the lipid moieties of glycosylphosphatidylinositol (GPI) group, which is present in some membrane proteins, might interfere with the capturing step of the technique. To find an answer, we evaluated the chemical denaturation and also the refolding behavior of insoluble and soluble alkaline phosohatase (ALP), with or without GPI group, respectively. The results indicated that the presence of GPI in the enzyme increased the stability of the protein against chemical denaturation while it decreased its refolding yield by the artificial chaperone refolding technique. The lower refolding yield, compared to soluble ALP (sALP), might be due to a less efficient stripping step caused by new interactions imparted to the refolding elements of the system especially those among the hydrophobic tails of GPI and the capturing agent of the technique. These new interactions will interrupt the kinetics of detergent stripping from the captured molecules by the stripping agent (i.e., cyclodextrins). This situation will lead to higher intermolecular hydrophobic interactions among the refolding protein intermediates leading to their higher misfolding and aggregation.     

Modulation of Notch Signaling Pathway to Prevent H2O2/Menadione-Induced SK-N-MC Cells Death by EUK134

The brain in Alzheimer’s disease is under increased oxidative stress, and this may have a role in the pathogenesis and neural death in this disorder. It has been verified that numerous signaling pathways involved in neurodegenerative disorders are activated in response to reactive oxygen species (ROS). EUK134, a synthetic salen–manganese antioxidant complex, has been found to possess many interesting pharmacological activities awaiting exploration. The present study is to characterize the role of Notch signaling in apoptotic cell death of SK-N-MC cells. The cells were treated with hydrogen peroxide (H₂O₂) or menadione to induce oxidative stress. The free-radical scavenging capabilities of EUK134 were studied through the MTT assay, glutathione peroxidase (GPx) enzyme activity assay, and glutathione (GSH) Levels. The extents of lipid peroxidation, protein carbonyl formation, and intracellular ROS levels, as markers of oxidative stress, were also studied. Our results showed that H₂O₂/menadione reduced GSH levels and GPx activity. However, EUK134 protected cells against ROS-induced cell death by down-regulation of lipid peroxidation and protein carbonyl formation as well as restoration of antioxidant enzymes activity. ROS induced apoptosis and increased NICD and HES1 expression. Inhibition of NICD production proved that Notch signaling is involved in apoptosis through p53 activation. Moreover, H₂O₂/menadione led to Numb protein down-regulation which upon EUK134 pretreatment, its level increased and subsequently prevented Notch pathway activation. We indicated that EUK134 can be a promising candidate in designing natural-based drugs for ROS-induced neurodegenerative diseases. Collectively, ROS activated Notch signaling in SK-N-MC cells leading to cell apoptosis.     

Diversity of the rotifer Brachionus plicatilis species complex (Rotifera: Monogononta) in Iran through integrative taxonomy

Faunistic survey using a DNA taxonomy approach may provide different results from morphological methods, especially for small and understudied animals. In this study, we report the results from morphometric analyses (linear measurements of the lorica) and DNA taxonomy (generalized mixed Yule coalescent model on the barcoding mtDNA locus cytochrome c oxidase subunit I) performed on 15 clonal lineages of the rotifer Brachionus plicatilis species complex from six Iranian inland saltwaters. The DNA taxonomy approach found more units of diversity (four) than the morphometric approach (two) in the studied rotifers. Three of the taxa identified in this study are already known as described valid species or as‐yet unnamed lineages, but a new, additional lineage is also identified from Iran. © 2014 The Linnean Society of London     

Probing the active-site requirements of human intestinal N-terminal maltase glucoamylase: The effect of replacing the sulfate moiety by a methyl ether in ponkoranol,a naturally occurring α-glucosidase inhibitor

Ponkoranol is a naturally occurring glucosidase inhibitor isolated from the plant Salacia reticulata. The compound comprises a sulfonium ion with an internal sulfate counter ion. We report here an efficient synthetic route to 3′-O-methyl ponkoranol to test the hypothesis that occupation of a hydrophobic pocket by a methyl group instead of the polar sulfate ion within the active site of human N-terminal maltase glucoamylase would be beneficial. The synthetic strategy relies on the nucleophilic attack of 2,3,5-tri-O-benzyl-1,4-anhydro-4-thio-d-arabinitol at the C-6 position of benzyl 6-O-p-toluenesulfonyl β-d-glucopyranoside, followed by deprotection using boron trichloride and reduction with sodium borohydride. The target compound inhibited the N-terminal catalytic domain of intestinal human maltase glucoamylase (ntMGAM) with a K_i value of 0.50 ± 0.04 μM, higher than those of de-O-sulfonated ponkoranol (K_i = 43 ± 3 nM), or its 5′-stereoisomer (K_i = 15 ± 1 nM). We conclude that the interaction of the methyl group with hydrophobic residues in the active site is not as beneficial to inhibition of ntMGAM as the other interactions of the polyhydroxylated chain with active-site residues.     

Design,synthesis and biological evaluation of new 2,3-diarylquinoline derivatives as selective cyclooxygenase-2 inhibitors

A new group of 2,3-diarylquinoline derivatives possessing a methylsulfonyl COX-2 pharmacophore at the para-position of the C-2 phenyl ring were synthesized and evaluated as selective COX-2 inhibitors. In vitro COX-1/COX-2 structure–activity relationships were determined by varying the substituents on the C-4 quinoline ring. Among the 2,3-diarylquinolines, 2-(4-(methylsulfonyl) phenyl)-3-phenylquinoline-4-carboxylic acid (8) exhibited the highest potency and selectivity for COX-2 inhibitory activity (COX-2 IC₅₀ = 0.07 μM; selectivity index = 687.1) that was more selective than the reference drug celecoxib (COX-2 IC₅₀ = 0.06 μM; selectivity index = 405). A molecular modeling study where 8 was docked in the binding site of COX-2 indicated that the p-MeSO₂ COX-2 pharmacophore group on the C-2 phenyl ring is oriented in the vicinity of the COX-2 secondary pocket (Arg⁵¹³, Phe⁵¹⁸ and Val⁵²³) and the carboxylic acid substituent can interact with Ser⁵³⁰. The structure activity data acquired indicate that the size and nature of the C-4 quinoline substituent are important for COX-2 inhibitory activity.     

Kinetic aspects of alkaline phosphatase refolding in the presence of alpha-cyclodextrin

To get a better understanding of the molecular aspects of protein folding, the refolding kinetic behavior of guanidine hydrochloride-denatured alkaline phosphatase (ALP) was studied in the presence of alpha-cyclodextrin (alpha-CD) through two different approaches: the dilution additive and the artificial chaperone-assisted methods. It was found that alpha-CD enhanced the recovered activity more than 50% via both approaches while decreased the refolding rate, perhaps due to engaging the hydrophobic patches of the protein in a rigid conformation. In contrast, detergents used in the artificial chaperone method increased the refolding rate significantly. A comparison of the rate constants for the refolding and the activity recovery of denatured ALP in the presence of various concentrations of CD and different kinds of detergents showed that they do not progress in a synchronized pattern. This may be attributed to continuous structural rearrangements in the protein long after the return of enzyme activity. These observations are discussed in terms of kinetic and structural aspects of the refolding pathway.     

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.