Seiichi Morita

Protein refolding using stimuli-responsive polymer-modified aqueous two-phase systems

The function of a stimuli-responsive polymer was studied for the utilization of protein unfolding and refolding in protein separation using aqueous two-phase systems (ATPS). Poly(ethylene glycol) (PEG) bound to a thermo-reactive hydrophobic head (poly(propylene oxide)-phenyl group (PPO-Ph group)) was used as the functional ligand to modify the PEG phase of the aqueous two-phase systems. Firstly, refolding of carbonic anhydrase from bovine (CAB) was examined in the presence of PPO-Ph-PEG at various temperatures. The refolding yield of CAB was strongly enhanced and aggregate formation was suppressed by addition of PPO-Ph-PEG at a specific temperature (50-55 degrees C). The change in the local hydrophobicity of CAB and PPO-Ph-PEG was characterized using the aqueous two-phase partitioning method and a hydrophobic fluorescent probe. The local hydrophobicity of CAB was maximized at 60 degrees C. The local hydrophobicity of PPO-Ph-PEO was also found to be increased above 45 degrees C. A simple model for CAB refolding, which includes (i) PPO-Ph-PEG complex formation and CAB in the intermediate state and (ii) refolding and release of native CAB from the PPO-Ph-PEG surface, is suggested based on the evaluated surface hydrophobicity.

Novel array-type gas sensors using conducting polymers, and their performance for gas identification

Novel gas sensor devices have been developed using polythiophene (pTh) film and poly(3-n-dodecylthiophene) (pDpTh) film coated over pTh film. These polymer films were electrochemically deposited and doped by cyclic voltammetry on thin-film electrodes where the isolation gap was formed by a micromachining process. We examined the response characteristics of the conducting polymer films against various sample gases over a range of temperatures of the sensitive layer. The resistance changes of both sensitive layers of pTh and pDpTh were highly dependent on the kind of layer. In particular, pTh film responded to ammonia gas and pDpTh films clearly responded to hydrophobic gases, such as chloroform, methane and ethanol. The response of these films to several gases was analyzed with a pattern recognition (PARC) algorithm. It was found that our simple gas sensor device could discriminate between the gases that were used here.

Detection of a heat stress-mediated interaction between protein and phospholipid membrane using dielectric measurement

The dielectric response of lipid bilayer membrane vesicles (liposomes) prepared using either phosphatidylcholine from egg (EPC) or 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) was analyzed at a frequency range of 0.1 to 100 MHz. A marked dielectric dispersion for EPC and POPC liposome suspensions was observed above 1 MHz. An appropriate analysis of the dielectric dispersion curve was performed using the Cole-Cole equation and the Debye equation and was found to provide a method for the determination of dielectric parameters. Among the dielectric parameters, the characteristic frequency of a second dispersion around 50 MHz varied corresponding with changes in the test conditions. Of particular note is that an anomalous change in the characteristic frequency in the presence of protein corresponded to the degree of hydrophobic interaction between proteins and liposomes. The value of the frequency around 50 MHz, as well as the decrease in permittivity over the frequency range tested, are indicators of the interaction between proteins and liposomes.

Systematical Characterization of Phase Behaviors and Membrane Properties of Fatty Acid/Didecyldimethylammonium Bromide Vesicles

Fatty acids (FAs) are known to form vesicle structures, depending on the surrounding pH conditions. In this study, we prepared vesicles by mixing FAs and a cationic surfactant, and then investigated their physicochemical properties using fluorescence spectroscopy and dielectric dispersion analysis (DDA). The assemblies formed from oleic acid (OA) and linoleic acid (LA) were modified by adding didecyldimethylammonium bromide (DDAB). The phase state of FA/DDAB mixtures was investigated with pH titration curves and turbidity measurements. The trigonal diagram of FA/ionized FA/DDAB was successfully drawn to understand the phase behaviors of FA/DDAB systems. The analysis of fluidities in the interior of the membrane with use of 1,6-diphenyl-1,3,5-hexatriene (DPH) indicated that the membrane fluidities of OA/DDAB and LA/DDAB at pH 8.5 slightly decreased in proportion to the molar ratio of DDAB in FA/DDAB systems. The fluorescent probe 6-lauroyl-2-dimethylamino naphthalene (Laurdan) indicated that the LA vesicle possessed a dehydrated surface, while the OA vesicle surface was hydrated. Modification of LA vesicles with DDAB induced the hydration of membrane surfaces, whereas modification of OA vesicles by DDAB had the opposite effect. DDA analysis indicated that the membrane surfaces were hydrated in the presence of DDAB, suggesting that the surface properties of FA vesicles are tunable by DDAB modification.

Stimuli-responsive separation of proteins using immobilized liposome chromatography

The possibility of the stimuli-responsive separation of proteins was investigated using immobilized liposome chromatography (ILC) as novel aqueous two-phase systems. The specific capacity factor (k(s)) of beta-galactosidase, obtained by analysis of ILC, was varied by changing the pH of the solution and was maximized at the specific pH of 5 (k(s),max = 5.57). The k(s) values were found to correspond well with their local hydrophobicities, which can be determined by the aqueous two-phase partitioning method. The variation of k(s), therefore, indicates a change in the surface properties of a protein during conformational change under pH stimuli. A similar phenomenon is observed in the case of other proteins (alpha-glucosidase, k(s),max = 11.3 at pH 4; carbonic anhydrase from bovine, k(s),max = 6.53 at pH 4). The difference in the height and/or the position of the peaks of the ks-pH curves of each protein suggests a difference in their pH denaturation in the ILC column. Based on these results, the mutual separation of the above proteins at pH 4 could be successfully performed by selecting their specific capacity factor as a design parameter.

Dielectric response of cells and liposomes and its utilization for evaluation of cell membrane-protein interaction

The dielectric measurements of an Escherichia coli (E. coli) cell suspension and liposome suspensions were carried out in the frequency range between 0.1 and 100 MHz to detect the heat stress-mediated interaction between proteins and cell membranes. The dielectric relaxation dispersion was observed to be above 1 MHz. The dielectric parameter (amplitude of dispersion, deltaepsilon) based on the Cole-Cole equation was anomalously changed with increasing temperature. The value of deltaepsilon of liposomes was varied at various temperatures depending on the type of protein present. The change in deltaepsilon of liposomes correlated with the amount of protein translocated across the phospholipid membrane. The changes in the value of deltaepsilon of E. coli cells with temperature variation was similar to those of liposomes in the presence of proteins, suggesting that the variation in dielectric parameters reflected the interaction between the phospholipid membrane and proteins. It was found that the dielectric measurement could be utilized for the detection of the interaction between proteins and liposomes.

Characterization and on-line monitoring of cell disruption and lysis using dielectric measurement.

The dielectric measurement of Escherichia coli suspension was carried out between 0.01 and 100 MHz during cell disruption using a physical method or cell lysis induced by chemicals for the purpose of on-line monitoring of cell disruption or lysis of bacterial cells. The dielectric dispersion of relative permittivity centered at about 1.4 MHz and the dielectric parameters based on the fitting calculation well reflected the damage of these cells under physical and chemical stresses. The degree of cell disruption as determined by the dielectric parameters, the amplitude of dispersion and the conductivity, well corresponded to those obtained by other conventional kinetic analyses. This methodology can be utilized for the on-line monitoring of physical cell disruption and cell lysis induced by detergent and could provide direct information to control these processes. Based on these results, the dielectric measurement was successfully applied to monitor the stress-mediated cultivation of E. coli cells.