Adsorptive refolding of a highly disulfide-bonded inclusion body protein using anion-exchange chromatography

α-Fetoprotein (AFP) is a prospective biopharmaceutical candidate currently undergoing advanced-stage clinical trials for autoimmune indications. The high AFP expression yields in the form of inclusion bodies in Escherichia coli renders the inclusion body route potentially advantageous for process scale commercial manufacture, if high-throughput refolding can be achieved. This study reports the successful development of an ‘anion-exchange chromatography’-based refolding process for recombinant human AFP (rhAFP), which carries the challenges of contaminant spectrum and molecule complexity. rhAFP was readily refolded on-column at rhAFP concentrations unachievable with dilution refolding due to viscosity and solubility constraints. DEAE-FF functioned as a refolding enhancer to achieve rhAFP refolding yield of 28% and product purity of 95% in 3 h, at 1 mg/ml protein refolding concentration. Optimization of both refolding and chromatography column operation parameters (i.e. resin chemistry, column geometry, redox potential and feed conditioning) significantly improved rhAFP refolding efficiency. Compared to dilution refolding, on-column rhAFP refolding productivity was 9-fold higher, while that of off-column refolding was more than an order of magnitude higher. Successful demonstration that a simple anion-exchange column can, in a single step, readily refold and purify semi-crude rhAFP comprising 16 disulfide bonds, will certainly extend the application of column refolding to a myriad of complex industrial inclusion body proteins.     

Molecular chemistry in a zeolite: genesis of a zeolite Y-supported ruthenium complex catalyst

Dealuminated zeolite Y was used as a crystalline support for a mononuclear ruthenium complex synthesized from cis-Ru(acac)2(C2H4)2. Infrared (IR) and extended X-ray absorption fine structure spectra indicated that the surface species were mononuclear ruthenium complexes, Ru(acac)(C2H4)2(2+), tightly bonded to the surface by two Ru-O bonds at Al(3+) sites of the zeolite. The maximum loading of the anchored ruthenium complexes was one complex per two Al(3+) sites; at higher loadings, some of the cis-Ru(acac)2(C2H4)2 was physisorbed. In the presence of ethylene and H2, the surface-bound species entered into a catalytic cycle for ethylene dimerization and operated stably. IR data showed that at the start of the catalytic reaction, the acac ligand of the Ru(acac)(C2H4)2(2+) species was dissociated and captured by an Al(3+) site. Ethylene dimerization proceeded approximately 600 times faster with a cofeed of ethylene and H2 than without H2. These results provide evidence of the importance of the cooperation of the Al(3+) sites in the zeolite and the H2 in the feed for the genesis of the catalytically active species. The results presented here demonstrate the usefulness of dealuminated zeolite Y as a nearly uniform support that allows precise synthesis of supported catalysts and detailed elucidation of their structures.     

Use of block copolymer as compatibilizer in polyimide/zeolite composite membranes

In this work, we introduced a diblock copolymer (dBC), i.e., polystyrene‐b‐poly(hydroxyl ethyl acrylate) (PS‐b‐PHEA) as a compatibilizer to enhance interfacial adhesion between PI and zeolite in PI/Zeolite/dBC (1/0.1/0.05 wt%) membrane for gas separation. FT‐IR spectroscopy showed the formation of hydrogen bonding interactions of the carbonyl and the hydroxyl in dBC with both PI and zeolite. The differential scanning calorimeter (DSC) study showed that the glass transition temperature (T_g) of PI increased upon the introduction of dBC, indicating specific interactions in the mixed matrix membranes. The gas permeabilities of H₂, N₂, O₂, and CO₂ through PI/zeolite 5A/dBC membranes were reduced but the permselectivity were increased compared to neat PI membrane. Copyright © 2009 John Wiley & Sons, Ltd.     

Rubidium doped zeolite rho: structure and microwave conductivity of a metallic zeolite

Large crystallites of high purity zeolite rho were synthesized by controlled monitoring of the aging and heating period of the mother gel. The microwave conductivity of Rb-rho doped with up to 20 Rb atoms per unit cell was measured over the temperature range 15-300 K, and the structures of three of the samples were examined through Rietveld analysis of powder neutron diffraction data. At low concentrations of rubidium dopant the observed microwave responses were dominated by polarization effects. In the sample Rb(17)/Rb-rho a strongly temperature-dependent electronic contribution to the conductivity was observed above approximately =150 K. In Rb(20)/Rb-rho, conductivities in the range 1.5-2.3 Sm(-1) were observed between 15 and 300 K. This residual conductivity at 15 K, unprecedented in a zeolite, indicates that the sample is indeed metallic; however, the values of conductivity measured are low in comparison to conventional metals and comparable to those of doped semiconductors. The evolution of the conducting behaviour is discussed in relation both to observed structural and to possible electronic changes occurring within the samples on metal doping.     

Use of diffusion barriers in the preparation of supported zeolite ZSM-5 membranes

Zeolite ZSM-5 membranes were prepared by in situ crystallization on porous α-Al₂O₃ disks that contained a diffusion barrier to limit excessive penetration of siliceous species into the alumina pores. The barrier was introduced into the alumina pores by impregnating the porous disk with a 1:1 molar mixture of furfuryl alcohol (FA) and tetraethylorthosilicate (TEOS), polymerizing the mixture retained in the disk, and carbonizing the resulting polymer at 600°C in N₂. Following carbonization, a partial carbon burn-off was carried out by catalyzed oxidation in 2% O₂N₂ at 600°C to generate a carbon-free region near the surface of the support. After zeolite crystallization, the remaining carbon and the organic structure directing agent were removed by calcination in air, at 500°C. It was found that pure carbon does not support zeolite growth while the solid obtained from a mixture of FA and TEOS does, due to the presence of dispersed silica. Membranes synthesized using barriers have n-butane flux and n-butane/isobutane selectivity 2.7 × 10⁻³ mol m⁻² s⁻¹ and 45, respectively, at 185°C, which are, respectively, ca. 1.6 and 4 times as large as those of membranes prepared without the use of barriers. Electron probe microanalysis (EPMA) and X-ray diffraction (XRD) revealed that the internal layer of the barrier-pretreated membrane has smaller thickness and higher crystallinity accounting for the increased flux and selectivity.     

SSZ-60: a new large-pore zeolite related to ZSM-23

SSZ-60 is a new borosilicate zeolite that may be prepared using N-ethyl-N-(2,4,4-trimethylcyclopentyl)pyrrolidinium or N-ethyl-N-(3,3,5-trimethylcyclohexyl)pyrrolidinium as a structure directing agent; the framework of SSZ-60 was determined by the FOCUS Fourier recycling method, its structure refined in space group P21nm and found to possess a one-dimensional channel system with pores delimited by twelve-rings; the topological structure of SSZ-60 may be derived from a sigma-expansion of the framework of ZSM-23 (MTT).     

Use of photolithography to encode cell adhesive domains into protein microarrays

Protein microarrays are rapidly emerging as valuable tools in creating combinatorial cell culture systems where inducers of cellular differentiation can be identified in a rapid and multiplexed fashion. In the present study, protein microarraying was combined with photoresist lithography to enable printing of extracellular matrix (ECM) protein arrays while precisely controlling "on-the-spot" cell-cell interactions. In this surface engineering approach, the micropatterned photoresist layer formed on a glass substrate served as a temporary stencil during the microarray printing, defining the micrometer-scale dimensions and the geometry of the cell-adhesion domains within the printed protein spots. After removal of the photoresist, the glass substrates contained micrometer-scale cell-adhesive regions that were encoded within 300 or 500 microm diameter protein domains. Fluorescence microscopy and atomic force microscopy (AFM) were employed to characterize protein micropatterns. When incubated with micropatterned surfaces, hepatic (HepG2) cells attached on 300 or 500 mum diameter protein spots; however, the extent of cell-cell contacts within each spot varied in accordance with dimensions of the photoresist stencil, from single cells attaching on 30 microm diameter features to multicell clusters residing on 100 or 200 microm diameter regions. Importantly, the photoresist removal process was shown to have no detrimental effects on the ability of several ECM proteins (collagens I, II, and IV and laminin) to support functional hepatic cultures. The micropatterning approach described here allows for a small cell population seeded onto a single cell culture substrate to be exposed to multiple scenarios of cell-cell and cell-surface interactions in parallel. This technology will be particularly useful for high-throughput screening of biological stimuli required for tissue specification of stem cells or for maintenance of differentiated phenotype in scarce primary cells.     

Use of organic solvent to prevent protein adsorption in CE-MS experiments

Protein adsorption onto capillary wall often hampers CE separations, particularly in the CZE mode. Electrostatic interactions are not the only factors affecting adsorption, as hydrophobic interactions and/or protein conformational changes are also involved in the adsorption phenomenon. Numerous methods can be used to reduce or avoid adsorption, such as (i) addition of low molecular weight molecules in the BGE, (ii) use of surfactants, or (iii) capillary coatings. However, most of these methods are not MS-compatible. In this study, we evaluated the addition of organic solvent as an alternative MS-compatible method to decrease protein adsorption. The effect of the solvent addition was emphasized using classical methods for estimating reversible and irreversible adsorption. In many cases, organic solvents were effective at decreasing adsorption. However, the influence of the organic solvent on protein adsorption should be evaluated case-by-case in CE method development.     

Use of a novel Hill-climbing genetic algorithm in protein folding simulations

We have developed a novel Hill-climbing genetic algorithm (GA) for simulation of protein folding. The program (written in C) builds a set of Cartesian points to represent an unfolded polypeptide’s backbone. The dihedral angles determining the chain’s configuration are stored in an array of chromosome structures that is copied and then mutated. The fitness of the mutated chain’s configuration is determined by its radius of gyration.

A four-helix bundle was used to optimise simulation conditions, and the program was compared with other, larger, genetic algorithms on a variety of structures. The program ran 50% faster than other GA programs. Overall, tests on 100 non-redundant structures gave comparable results to other genetic algorithms, with the Hill-climbing program running from between 20 and 50% faster. Examples including crambin, cytochrome c, cytochrome B and hemerythrin gave good secondary structure fits with overall alpha carbon atom rms deviations of between 5 and 5.6 Å with an optimised hydrophobic term in the fitness function.     

Use of granulated modified zeolite Y for the removal of inorganic arsenic and selenium species

Column studies were performed to evaluate the performance of modified zeolite-Y with ion Fe (zeolite-FeY) in removing As(III), As(V), Se(IV) and Se(VI) from groundwater. The removal capacities for zeolite-FeY was carried out on arsenic and selenium species in aqueous solution by co-precipitation technique with DBDTC-Pp complex in the pH range of 1.5–2.5 followed by the neutron activation analysis (NAA) using a TRIGA-MkII reactor with an average flux of 2.1 × 10¹² neutrons cm⁻² s⁻¹ and inductively coupled plasma-mass spectrometry (ICP-MS) technique as comparison. The accuracy between the results obtained from both techniques were compared and evaluated.     

Silver nitrate in silver zeolite A: three-dimensional incommensurate guest ordering in a zeolite framework

We report the results of a detailed examination of the occlusion of silver nitrate in silver zeolite A (AgA). The superlattice reported to occur in (AgNO3)9-AgA was found to melt at between 80 and 100 degrees C on heating and reappear when the sample was cooled down to 80 degrees C. Annealing in this temperature range and rigorous exclusion of water produced an enhancement of the superlattice peaks, which results from ordering of the contents of the zeolite cages. Peaks assigned to the superlattice were indexed with the tetragonal lattice parameters a = 17.440(5) and c = 12.398(4) A and proposed space group P4/nmm. The sharp peaks representing the lattice of the framework (a = 12.3711(5) A, Pm3m) remained largely unaffected by the guest in this compound, which was found to exhibit strong negative thermal expansion. The host and guest lattices are incommensurate with the tetragonal guest lattice being slightly larger than the cubic host in the c-direction and slightly smaller in the a- and b-directions.     

Use of quasi-isoelectric buffers to limit protein adsorption in capillary zone electrophoresis

The use of quasi-isoelectric buffers consisting of narrow pH cuts of carrier ampholytes (NC) has been investigated to limit protein adsorption on capillary walls during capillary zone electrophoresis experiments. To quantify protein adsorption on the silica surface, a method derived from that of Towns and Regnier has been developed. alpha-Lactalbumin (14 kDa, pI 4.8) and alpha-chymotrypsinogen A (25 kDa, pI 9.2) have been used as model proteins. Acidic narrow pH cuts of carrier ampholytes (NC, pH 3.0) obtained from fractionation of Serva 4-9 carrier ampholytes were used as BGE in bare-silica capillaries, and allowed to decrease significantly protein adsorption, as compared to experiments performed with classical formate buffer. The use of NC as BGE appeared to be as efficient as the use of polydimethylacrylamide coating to prevent protein adsorption. This increase of protein recovery when using NC was attributed to the interaction of carrier ampholytes with the silica surface, leading to a shielding of the capillary wall.     

Hydrodewaxing studies over a pentasil ferrisilicate zeolite

The catalytic dewaxing of an atmospheric gas oil has been carried out over Pt-loaded catalysts containing a ferrisilicate zeolite of the ZSM-5 structure. It is observed that the mode of incorporation of the metallic function has a profound effect on the activity on the catalyst. Further, the use of alumina (instead of bentonite) as binder increases the activity of the catalyst.

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Heteroepitaxial growth of a zeolite film with a patterned surface-texture

We focus on the eteroepitaxial growth of trigonal chabazite with voids of three-dimensional intersection structures. The differences in the structures originate from the stacking sequences of the six-membered rings. Sodalite along 111 is constructed by an "abcabc" stacking sequence, while chabazite along [111] is constructed by "abab". Therefore, chabazite can grow heteroepitaxially on a sodalite substrate while keeping the relation of chabazite (111)//sodalite {111}. In this Communication, we report on the first success of the heteroepitaxial growth of a continuous zeolite (chabazite) film with a unique patterned surface-texture on a millimeter-sized sodalite single crystal. We have found that the unique texture of chabazite films is rationalized by the heteroepitaxial growth of chabazite on sodalite and the subsequent twin formation of chabazite.