The Inducible Intein-Mediated Self-Cleaving Tag (IIST) System: A Novel Purification and Amidation System for Peptides and Proteins

An efficient self-cleavable purification tag could be a powerful tool for purifying recombinant proteins and peptides without additional proteolytic processes using specific proteases. Thus, the intein-mediated self-cleavage tag was developed and has been commercially available as the IMPACT™ system. However, uncontrolled cleavages of the purification tag by the inteins in the IMPACT™ system have been reported, thereby reducing final yields. Therefore, controlling the protein-splicing activity of inteins has become critical. Here we utilized conditional protein splicing by salt conditions. We developed the inducible intein-mediated self-cleaving tag (IIST) system based on salt-inducible protein splicing of the MCM2 intein from the extremely halophilic archaeon, Halorhabdus utahensis and applied it to small peptides. Moreover, we described a method for the amidation using the same IIST system and demonstrated ¹⁵N-labeling of the C-terminal amide group of a single domain antibody (V_HH).     

Effective Mutual Separation of Europium(Ⅲ) from Gadolinium(Ⅲ) by Using ODS Modified with Bis(l,l,3,3-tetramethylbutyl) phosphinic acid——Extraction Chromatography in HGlyc Acid-HN03 System

It is well known that lanthanides is one of the most difficult groups of elements to be separated in the periodical table because of the so-called lanthanide contraction. Among lanthanides,the 4f orbital of gadolinium(Ⅲ) is half-filled——4f⁷,such a stable configuration leads to the effects from coordination field weaker on gadolinium than that on the adjacent lanthanides, and the stabilities of gadolinium(Ⅲ) complexes somewhat lower than those of europium(Ⅲ) in some cases.     

Misleading fringes in TEM images and diffraction patterns of Si nanocrystallites

High‐resolution transmission electron microscopy (HRTEM) images and electron diffraction patterns of twinned Si nanocrystallites were recorded along various directions and analyzed in detail. We point out that special attention must be paid when interpreting HRTEM images and diffraction patterns of twinned Si nanocrystallites, because elongation of reciprocal lattice points could fabricate misleading fringes and patterns. (© 2003 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)     

Preparation of poly(2,4-dichloro-6-vinyl-1,3,5-triazine-co-styrene)s as polymer supports containing two replaceable groups in monomer unit

Poly(2,4-dichloro-6-vinyl-1,3,5-triazine-co-styrene)s were prepared by the reaction of poly(2,4-diamino-6-vinyl-1,3,5-triazine-co-styrene)s with hydrochloric acid, and followed by treatment with thionyl chloride and N,N-dimethylformamide. The chlorine atoms in the resulted polymers were replaced readily by several nucleophiles such as amide, alkoxide, and mercaptide to afford the corresponding polymers in moderate yields. Among these, polymers containing oligo (oxyethylene) groups at both 2 and 4 positions of 1,3,5-triazine ring worked effectively as phase transfer catalysts for the reaction of n-octyl bromide with alkali metal thiocyanates in toluene–water systems. However, polystyrenes containing only one oligo (oxyethylene) group in monomer unit scarcely exhibited the activity under these conditions although the degree of loading of the functional group was almost the same. The activity depended on the number of oxyethylene units, and the selectivity to alkali metal ions was also observed. © 1992 John Wiley & Sons, Inc.     

Effect of strain in the barrier layer on structural and optical properties of highly strained In0.77Ga0.23As/InGaAs multiple quantum wells

We have investigated the effect of the barrier strain in +1.65%-strained In_0.77Ga_0.23As/InGaAs multiple quantum wells (MQWs) on the structural and optical properties by means of double-crystal X-ray diffraction, transmission electron microscopy (TEM), and room-temperature photoluminescence (PL). The optimum condition of the barrier layer deduced from the X-ray and the PL measurements was nearly lattice-matching, i.e., strain from −0.40 to +0.20% is required for the sharp X-ray diffraction satellite peaks and from −0.17 to +0.14% for large PL intensity. Under compressive strain in the barrier layer, misfit dislocations are introduced into the MQW structures. In the case of tensile strain, however, threading dislocations originating from the thickness undulations in the wells and the barriers are observed. The TEM studies reveal that the thickness undulations are induced by the compositional modulation. The undulation and modulation are enhanced by increasing the tensile strain in the barrier layers. These results indicate that the strain-compensation does not work well on the MQW containing such highly strained InGaAs wells.     

Immobilization of Photosynthetic Pigments into Silica-Surfactant Nanocomposite Films

Highly transparent silica-surfactant nanocomposite films containing photosynthetic pigments have been successfully formed through the solubilization of chlorophyll a (Chl a) into surfactant micelles. The UV-vis absorption spectra indicated that a large amount of Chl a were transformed into pheophytin a in the films. These photosynthetic pigments were well dispersed in the surfactant assemblies and their chlorin rings were exposed to the surface of silica layers. Even under an air atmosphere, the photostability of immobilized pigments was largely improved in comparison with that in a homogeneous Chl a solution. Because both Chl a and pheophytin a molecules are effective for the photosensitive charge separation, the present film system is very suitable for heterogeneous immobilizing media for photosynthetic pigments from the viewpoint of in vitro biomimetic devices for solar energy conversion.     

A Liquid Chromatography-Mass Spectrometry Method to Study the Interaction between Membrane Proteins and Low-Molecular-Weight Compound Mixtures

Molecular interaction analysis is an essential technique for the study of biomolecular functions and the development of new drugs. Most current methods generally require manipulation to immobilize or label molecules, and require advance identification of at least one of the two molecules in the reaction. In this study, we succeeded in detecting the interaction of low-molecular-weight (LMW) compounds with a membrane protein mixture derived from cultured cells expressing target membrane proteins by using the size exclusion chromatography-mass spectrometry (SEC-MS) method under the condition of 0.001% lauryl maltose neopentyl glycol as detergent and atmospheric pressure chemical ionization. This method allowed us to analyze the interaction of a mixture of medicinal herbal ingredients with a mixture of membrane proteins to identify the two interacting ingredients. As it does not require specialized equipment (e.g., a two-dimensional liquid chromatography system), this SEC-MS method enables the analysis of interactions between LMW compounds and relatively high-expressed membrane proteins without immobilization or derivatization of the molecules.