Cell surface protein engineering facilitated by accumulation of information on genome and protein structure involves heterologous production and modification of cell surface proteins using genetic engineering, and is important for the development of high-performance whole-cell catalysts. In this field, cell surface display is a major technology by exposing target proteins, such as enzymes, on the cell surface using a carrier protein. The target proteins are fused to the carrier proteins that transport and tether them to the cell surface, as well as to a secretion signal. This paper reviews cell surface display systems for prokaryotic and eukaryotic cells from the perspective of carrier proteins, which determine the number of displayed molecules, and the localization, size, and direction (N- or C-terminal anchoring) of the passengers. We also discuss advanced methods for displaying multiple enzymes and a new method for the immobilization of whole-cell catalysts using adhesive surface proteins.
We developed a fully automated electrophoresis system for rapid and highly reproducible protein analysis. All the two-dimensional (2D) electrophoresis procedures including isoelectric focusing (IEF), on-part protein staining, sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), and in situ protein detection were automatically completed. The system comprised Peltiert devices, high-voltage generating devices, electrodes, and three disposable polymethylmethacrylate (PMMA) parts for IEF, reaction chambers, and SDS-PAGE. Because of miniaturization of the IEF part, rapid IEF was achieved in 30 min. A gel with a tapered edge gel on the SDS-PAGE part realized a connection between the parts without use of a gluing material. A biaxial conveyer was employed for the part relocation, sample introduction, and washing processes to realize a low-maintenance and cost-effective automation system. Performances of the system and a commercial minigel system were compared in terms of detected number, resolution, and reproducibility of the protein spots. The system achieved high-resolution comparable to the minigel system despite shorter focusing time and smaller part dimensions. The resulting reproducibility was better or comparable to the performance of the minigel system. Complete 2D separation was achieved within 1.5 h. The system is practical, portable, and has automation capabilities. 相似文献
The neurotoxicity of the 42-mer and 40-mer amyloid beta peptides (Abeta42 and Abeta40) is closely related to the radicalization at both Tyr10 and Met35. Abeta42 is more neurotoxic than Abeta40. Our previous structural analyses of Abeta42 suggested that Tyr10 and Met35 are brought closer together by the turn at positions 22 and 23, and the S-oxidized radical cation at position 35, which is the ultimate toxic radical species, can be produced effectively through oxidation by the phenoxy radical at position 10. To verify this idea, their separation was measured by site-directed spin labeling (MTSSL) by using ESR spectroscopy. Among the three kinds of Abeta42 derivatives, which are doubly or singly spin-labeled at position 10 and 35, only 10,35-MTSSL-Abeta42 showed a clear dipole coupling in continuous-wave ESR; this suggests that the intramolecular spin labels at position 10 and 35 in Abeta42 are located within approximately 15 A. In contrast, 10,35-MTSSL-Abeta40 did not give such signals. The distance between Tyr10 and Met35 in 10,35-MTSSL-Abeta40, which was successfully measured by pulsed ESR spectroscopy was 30 A long. The difference in the distance between Abeta42 and Abeta40 could explain in part the stronger neurotoxicity of Abeta42 compared to Abeta40. 相似文献
The effect of low-molecular-weight polyphenols extracted from lychee (Oligonol) on metabolic syndrome characterized by abdominal obesity was examined. We performed a clinical trial for Oligonol conducted by randomized double-blind, placebo-controlled study. Eighteen (male, 14; female, 4) adult volunteers with abdominal circumference over 85 cm were enrolled and divided into two groups, Oligonol and placebo groups. All subjects took two capsules of Oligonol (50 mg/capsule) or placebo twice a day for 10 weeks. Physical and haematological examinations as well as a CT scan of the abdomen were carried out, before (control) and 10 weeks after Oligonol intake. Clinical parameters of body weight, abdominal circumference and visceral fat volume were significantly decreased in the Oligonol group compared to the control. Insulin resistance was improved by Oligonol in conjunction with elevation of serum adiponectin. These results suggest that Oligonol ameliorates metabolic syndrome by reducing visceral fat obesity. 相似文献
The glucosyl transfer reaction of kojibiose phosphorylase (KP; EC 2.4.1.230) was examined using glycerol or myo-inositol as an acceptor. In the case of glycerol, KP produced two main transfer products: saccharides A and B. The structure of saccharide A was O-alpha-D-glucopyranosyl-(1-->1)-glycerol and that of saccharide B was O-alpha-D-glucopyranosyl-(1-->2)-O-alpha-D-glucopyranosyl-(1-->1)-glycerol. These results show that KP transferred a glucose residue to the hydroxyl group at position 1 of glycerol. On the other hand, when myo-inositol was used as an acceptor, KP produced four transfer products: saccharides 1-4. The structures of saccharides 1 and 2 were O-alpha-D-glucopyranosyl-(1-->1)- and O-alpha-D-glucopyranosyl-(1-->5)-myo-inositol, respectively; those of saccharides 3 and 4 were O-alpha-D-glucopyranosyl-(1-->2)-O-alpha-D-glucopyranosyl-(1-->1)- and O-alpha-D-glucopyranosyl-(1-->2)-O-alpha-D-glucopyranosyl-(1-->5)-myo-inositol, respectively. KP transferred a glucose residue to the hydroxyl group at position 1 or 5 of myo-inositol. On the basis of the structures of their glucosyl transfer products, glycerol and myo-inositol were found to have a common structure with three hydroxyl groups corresponding to the hydroxyl group of the glucose molecule at positions 2, 3 and 4. The conformation of these three hydroxyl groups in the structure is equatorial. This structure is the substrate recognition site of KP. It has been suggested that KP strictly recognizes the structures of glycerol and myo-inositol, and catalyzes the transfer reaction of a glucose residue to the hydroxyl group at position 1 in glycerol, and at position 1 or 5 in myo-inositol, corresponding to position 2 in glucose. 相似文献
A newly developed real-time PCR assay rapidly quantifies the total bacterial numbers in contaminated ready-to-eat vegetables and fruits compared with the standard plate count method. Primers targeting the rpoB gene, which encodes for the beta subunit of the bacterial RNA polymerase and which is common to most bacterial species, was used instead of the 16S rRNA gene, which has multiple copies and varies among bacterial species. A primer pair specific for rpoB was confirmed to amplify rpoB in a wide range of bacterial species after we assessed 49 strains isolated from five kinds of fruits and vegetables. We purchased fruits and vegetables from retail shops and enumerated the bacteria associated with them by use of real-time PCR and compared this to the number found by the culture method. We found a high correlation between the threshold PCR cycle number when compared with the plate count culture number. The real-time PCR assay developed in this study can enumerate the dominant bacterial species in ready-to-eat fruits and vegetables. 相似文献
The 'colloidal platinum' stabilized with polyvinylpyrrolidone (Pt/PVP-colloid) was dispersed in hydrogen-rich water (HW; hydrogen concentration, 0.82 ppm; oxidation-reduction potential, -583 mV) or regular water (RW; <0.01 ppm, +218 mV). And we evaluated the antioxidant activity of Pt/PVP-colloid in HW or RW on 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical scavenging and improvement of blood fluidity under 2,2'-azobis (2-amidinopropane) dihydrochloride (AAPH)-induced oxidative stress. When applied with the 0.25-0.5 ppm Pt/PVP-colloid in RW or HW, the level of DPPH radicals decreased to 77.5-59.6% or 16.1-5.6%, in contrast to the level as high as 81.3% for HW alone, respectively, as measured by an electron spin resonance method. The horse blood, which was subjected to AAPH-induced oxidative stress, was incubated for 24 hr with RW or HW, and thereafter required 13.7 sec (100%) or 5.7 sec (42.3%) for passing through the micro-channels in a rheology equipment. When treated with 0.5-1.0 ppm Pt/PVP-colloid in RW or HW, the blood passage time in the micro-channels decreased dose-dependently to 9.7-7.3 sec (71.6-53.8%) or 4.3-1.3 sec (32.8-10.3%), and the rate of micro-channels clogged with erythrocyte aggregates decreased to 23.8-21.0% or 15.8-9.8%, respectively, from 42.8% for no addition of Pt/PVP. By scanning electron microscopy, AAPH-treated erythrocytes lost intact surface morphology on the membrane together with protrusions and without hollows, being indicative of impaired transforming ability, and the rate of erythrocyte agglutination was increased to 46.2%. When treated the horse blood with HW alone significantly decreased the rate of erythrocyte agglutination to 29.6%, whereas 1.0 ppm Pt/PVP-colloid in RW or HW decreased it to 24.1% or 21.1%, respectively. Thus, DPPH-radical-scavenging and erythrocyte-protecting effects of Pt/PVP-colloid in HW were superior to those of Pt/PVP-colloid in RW or Pt/PVP-free HW. The results could be mainly attributed to the enhanced antioxidant activity of Pt/PVP in HW, which may be due to captured-hydrogen on platinum. 相似文献