Understanding the Molecular Mechanisms of Polyphenol Inhibition of Amyloid ß Aggregation.

Alzheimer's disease (AD) is highly associated with self-aggregation of amyloid ß (Aß) proteins into fibrils. Inhibition of Aß aggregation by polyphenols is one of the major therapeutic strategies for AD. Among them, four polyphenols (brazilin, resveratrol, hematoxylin, and rosmarinic acid) have been reported to be effective at inhibiting Aß aggregation, but the inhibition mechanisms are still unclear. In this work, these four polyphenols were selected to explore their interactions with the Aß17-42 pentamer by molecular dynamics simulation. All four polyphenols can bind to the pentamer tightly but prefer different binding sites. Conversion of the ß-sheet to the random coil, fewer interchain hydrogen bonds, and weaker salt bridges were observed after binding. Interestingly, different Aß17-42 pentamer destabilizing mechanisms for resveratrol and hematoxylin were found. Resveratrol inserts into the hydrophobic core of the pentamer by forming hydrogen bonds with Asp23 and Lys28, while hematoxylin prefers to bind beside chain A of the pentamer, which leads to ß-sheet offset and dissociation of the ß1 sheet of chain E. This work reveals the interactions between the Aß17-42 pentamer and four polyphenols and discusses the relationship between inhibitor structures and their inhibition mechanisms, which also provides useful guidance for screening effective Aß aggregation inhibitors and drug design against AD.

Hematoxylin modulates tau-RD protein fibrillization and ameliorates Alzheimer's disease-like symptoms in a yeast model.

Alzheimer's disease (AD) is one of the most serious neurodegenerative diseases with no effective treatment options available. The formation of insoluble amyloid fibrils of the hyperphosphorylated tau protein is intimately associated with AD, hence the tau protein has been a key target for AD drug development. In this work, hematoxylin was discovered as a dual functional compound, that is, acting in the inhibition of repeat domain of tau (tau-RD) protein fibrillogenesis and remodeling of pre-formed tau-RD fibrils in vitro. Meanwhile, hematoxylin was able to reduce the accumulation of tau-RD aggregates in Saccharomyces cerevisiae. Experimental and computational studies indicated that hematoxylin directly interacts with tau-RD protein through hydrophobic forces, hydrogen bonds, π-cation interactions, and π-π stackings. In addition, cellular viability assays showed that hematoxylin greatly reduced cytotoxicity induced by tau-RD aggregates. In summary, hematoxylin might be a promising candidate for further development as a potential therapeutic drug for AD patients.

Surface-anchored microbial enzyme-responsive solid lipid nanoparticles enabling colonic budesonide release for ulcerative colitis treatment.

Colon-targeted oral drug delivery systems (CDDSs) are desirable for the treatment of ulcerative colitis (UC), which is a disease with high relapse and remission rates associated with immune system inflammation and dysregulation localized within the lining of the large bowel. However, the success of current available approaches used for colon-targeted therapy is limited. Budesonide (BUD) is a corticosteroid drug, and its rectal and oral formulations are used to treat UC, but the inconvenience of rectal administration and the systemic toxicity of oral administration restrict its long-term use. In this study, we designed and prepared colon-targeted solid lipid nanoparticles (SLNs) encapsulating BUD to treat UC by oral administration. A negatively charged surfactant (NaCS-C12) was synthesized to anchor cellulase-responsive layers consisting of polyelectrolyte complexes (PECs) formed by negatively charged NaCS and cationic chitosan onto the SLNs. The release rate and colon-specific release behavior of BUD could be easily modified by regulating the number of coated layers. We found that the two-layer BUD-loaded SLNs (SLN-BUD-2L) with a nanoscale particle size and negative zeta potential showed the designed colon-specific drug release profile in response to localized high cellulase activity. In addition, SLN-BUD-2L exhibited excellent anti-inflammatory activity in a dextran sulfate sodium (DSS)-induced colitis mouse model, suggesting its potential anti-UC applications.

Development of metformin hydrochloride loaded dissolving tablets with novel carboxymethylcellulose/poly-l-lysine/TPP complex.

Natural polymers like polysaccharides, polypeptides and their derivatives are broadly applied in drug delivery due to excellent biocompatibility and biodegradability. In this study, the dissolving tablets, formed with carboxymethylcellulose/poly-l-lysine/tripolyphosphate (CMC/PLL/TPP) complex, were prepared using metformin hydrochloride (MetHCl) as model drug. Confocal laser scanning microscopy observation manifested that FITC-labeled PLL interacted with CMC and formed a uniform interior microstructure. Scanning electron microscope images showed the drug-loaded tablets had well-formed shapes with smooth surfaces. MetHCl embedded interior the microstructures of the tablets and represented in a crystal form. Thermo-gravimetric analysis and differential scanning calorimetry indicated that the drug-loaded tablets had stable thermal properties with less moisture content (3.52%). Fourier transform infrared spectrometer confirmed that the CMC/PLL/TPP complex was fabricated via the electrostatic interactions between -NH3+, -COO- and -[P2O54-]- groups. The drug-loaded tablets had a high drug loading efficiency of 85.76% and drug encapsulation efficiency of 81.47%, and a shorter wetting time of 2.16 min in SSF (pH 6.8) and lower swelling ratio of 233.34%. The drug loaded in the samples could be released completely within 10 min in simulated saliva fluid (SSF pH 6.8), indicating a rapid drug release and dissolving profile in the environment, which could be developed for dissolving tablets.

Preparation of pH-responsive DOX-loaded chitosan nanoparticles using supercritical assisted atomization with an enhanced mixer.

Chemotherapeutics are used extensively in cancer and encapsulating the drug in nanoparticles is an effective method to enhance therapeutic efficacy and reduce side effect. In this work, DOX-loaded chitosan nanoparticles were prepared using supercritical fluid assisted atomization introduced by a hydrodynamic cavitation mixer (SAA-HCM) from aqueous solution. The influences of solution concentration, CO2/solution ratio, mixer pressure, chitosan/DOX ratio and chitosan molecular weight on particle morphologies and sizes were investigated in detail. Well defined spherical nanoparticles with average diameter ranging from 120 nm to 250 nm were obtained, and the loading efficiency was up to 90%. FT-IR result showed that the structure of DOX was not changed after the SAA-HCM process. Zeta potential of the nanoparticles was about +50 mV. The in vitro drug release behavior conducted in the media with pH of 4.5, 6.5 and 7.4 respectively showed strongly pH responsive. In vitro cytotoxicity profiles revealed that the activity of DOX was well maintained after loaded into chitosan nanoparticles. The SAA-HCM process was demonstrated to be a promising technique for one-step production of polymer/drug composite nanoparticles suitable for cancer drug delivery from aqueous solution.

Preparation of micrometric powders of parathyroid hormone (PTH1-34)-loaded chitosan oligosaccharide by supercritical fluid assisted atomization.

Parathyroid hormone (PTH1-34)-loaded dry powders were fabricated from aqueous solution for pulmonary administration using supercritical fluid assisted atomization introduced by a hydrodynamic cavitation mixer (SAA-HCM). Herein, chitosan oligosaccharide (CSO) was selected as a carrier in an effort to enhance transmucosal absorption of the drug. Well-defined, separated and spherical PTH(1-34)/CSO composite microparticles were obtained, and the particles size could be well controlled with narrow distribution. Aerodynamic performance was determined using next generation impactor (NGI), and the mass median aerodynamic diameter (MMAD) ranged strictly 1-5 µm range with fine particle fraction (FPF) up to 63.51%. The structural integrity of coprecipitated PTH(1-34) was validated by HPLC, FT-IR and circular dichroism, and a high loading efficiency up to 92.8% was obtained. TGA analyses revealed its thermal stability was preserved and XRD patterns showed amorphous structure of particles. The SAA-HCM process is proposed as a green technique for preparation of inhalable protein/polymer composite dry powders without use of any organic solvents.

Chromatographic separation of phenyllactic acid from crude broth using cryogels with dual functional groups.

Phenyllactic acid (PLA) is an important organic acid with wide antimicrobial activities against gram-positive and gram-negative bacteria and some fungi. This interesting compound can be synthesized by the microbial fermentation or the bioconversion using phenylpyruvic acid (PPA) as the key substrate and microorganisms as the whole-cell biocatalysts. However, the isolation of high-purity PLA with a high recovery from the crude fermentation or conversion broth is a challenging task. In this work, the separation of PLA from the crude conversion broth prepared by employing Lactobacillus buchneri cells as the whole-cell catalysts was achieved by the chromatography using the poly(hydroxyethyl methacrylate) (pHEMA)-based cryogel with a combination of anion-exchange and hydrophobic benzyl groups. The static adsorption behaviors of PLA under different salt concentrations and the adsorption capacities of PLA on the cryogel were measured experimentally. The chromatographic performance of PLA from the crude conversion broth was compared with that from the clarified broth. The results showed that the pHEMA-based cryogel has a high capacity of PLA, i.e., 14.64 mg mL-1 cryogel, and the adsorption of PLA was influenced by the salt concentration. By using deionized water as running buffer, PLA with a high purity of 97.6% was obtained with one step elution using 0.3 M NaCl as the elution solution with the recovery at the range of 80.2-90.8% from crude feedstock without any pretreatment at various flow velocities. These values were close to those obtained for the clarified broth, i.e., the purity of 98.4% and the recovery of 92.3% under the same chromatography conditions at 1 cm min-1. The cryogel was then applied to separate PLA from clarified feedstock, high purity (>96.7%) and recovery (>91.4%) of PLA were found with 20 cycles, which verified the selectivity and robustness of prepared pHEMA-VBTAC cryogel. Therefore, the chromatography using pHEMA-based cryogel with the dual functional groups is an effective approach for the isolation of PLA directly from the crude bioconversion broth and thus could be interesting in the separation and production of high-purity PLA in industry.

Formulation of insulin-loaded N-trimethyl chitosan microparticles with improved efficacy for inhalation by supercritical fluid assisted atomization.

Supercritical fluid assisted atomization introduced by a hydrodynamic cavitation mixer (SAA-HCM) was proposed as a green technique to fabricate insulin-loaded dry powders for inhalation administration. N-trimethyl chitosan (TMC), a polymeric mucoadhesive absorption enhancer, was synthesized and successfully micronized from aqueous solution using SAA-HCM. The prepared well-defined spherical TMC microparticles with preserved structure and thermal stability were potential carriers for delivery of proteins. Then, insulin-loaded TMC microparticles with high loading efficiency were coprecipitated from aqueous solutions using SAA-HCM without use of any organic solvents. The polymer/protein ratio revealed to be a factor influencing the particle morphology, and non-coalescing composite microparticles in amorphous state mainly ranging from 1µm to 5µm could be obtained in this work. Aerodynamic properties were assessed by next generation impactor (NGI) and the mass median aerodynamic diameter (MMAD) lied inside the inhalable range of 1-5µm, while fine particle fraction (FPF) reached above 60%. The structural integrity of encapsulated insulin was confirmed by HPLC, circular dichroism and fluorescence spectroscopy. In vivo study demonstrated that TMC could enhance the absorption and bioavailability of the pulmonarily administered insulin formulation for SD rats. These results suggest that TMC microparticles could be efficiently prepared as a promising vehicle for drug delivery, and SAA-HCM is a promising technique to prepare inhalable polymer/protein composite dry powders.

Supercritical fluid assisted production of micrometric powders of the labile trypsin and chitosan/trypsin composite microparticles.

Supercritical fluid assisted atomization introduced by a hydrodynamic cavitation mixer (SAA-HCM) was used to prepare micrometric particles of a labile protein, i.e., trypsin from aqueous solution without use of any organic solvents. The trypsin particles precipitated had various morphologies under different process conditions, with particle diameters ranging from 0.2 to 4 µm. FTIR, SDS-PAGE, CD and fluorescence spectra were performed to analyze the structural stability of the protein, and trypsin retained above 70% of the biological activity. Besides, chitosan was selected as the polymer carrier in an effort to prepare trypsin composite microparticles via SAA-HCM process. The influences of chitosan molecular weight, polymer/protein ratio and solution concentration on the particle morphology and size distribution were investigated in detail. Non-coalescing spherical composite microparticles with a narrow particle distribution (0.2-3 µm) could be obtained. The SAA-HCM prepared particles were amorphous as demonstrated by XRD and had a loading efficiency about 90%. The protein release profiles of the composite microparticles were evaluated using both the immersion condition and a Franz diffusion cell. Finally, the distribution of the protein within the particles was characterized through CLSM analysis of FITC-labeled trypsin-loaded chitosan microparticles. The SAA-HCM process is demonstrated to be a protein-friendly and promising technique for production of protein and polymer/protein composite particles formulations from aqueous solutions for drug delivery systems.

Chromatographic adsorption of serum albumin and antibody proteins in cryogels with benzyl-quaternary amine ligands.

The preparation and characterization of mixed-mode adsorbents for a typical separation purpose are of great importance in bioseparation areas. In this work, we prepared a new monolithic cryogel with a combination of ion-exchange and hydrophobic functions by employing benzyl-quaternary amine groups. The fundamental cryogel properties, protein equilibrium adsorption isotherm and chromatographic adsorption in the cryogel were measured experimentally. The results showed that, by using bovine serum album as the model protein, the dual functional cryogel has protein binding capability even in salt solution and the buffer with pH close or below the protein isoelectric point due to both the electrostatic and hydrophobic interactions. A capillary-based adsorption model was developed, which provided satisfied insights of the microstructure, axial dispersion, mass transfer as well as protein adsorption characteristics within the cryogel bed. The chromatographic isolation of bioactive proteins from rabbit blood serum was carried out by the cryogel. Immunoglobulin G antibody with a purity of 98.2% and albumin with a purity of 96.8% were obtained, indicating that the cryogel could be an interesting and promising adsorbent in bioseparation areas.

Co-expression of disulfide oxidoreductases DsbA/DsbC markedly enhanced soluble and functional expression of reteplase in Escherichia coli.

Reteplase is the third generation of thrombolytic medicine and has many advantages over commercial t-PA. However, over-expressing recombinant reteplase in E. coli always accumulates as inclusion bodies due to nine pairs of disulfide bonds formation that is the main obstacle for correct folding. In this paper, in order to enhance soluble expression of recombinant reteplase in E. coli, DsbA/DsbC foldases were used to introduce disulfide bonds into the reduced polypeptide chain and catalyze their isomerization to the native disulfide linkage during the folding process. Firstly multiple E. coli protein expression systems, i.e. DsbA, DsbC and DsbA/DsbC co-expression were constructed. Subsequently, IPTG and l-arabinose were added to induce expression of foldases and reteplase accordingly, and experimental parameters such as culture temperature and inducer concentration were optimized. As a result, the co-expression system markedly enhanced soluble expression of recombinant reteplase, and up to 60% of reteplase achieved soluble expression especially for the DsbC co-expression system. The fibrin plate method for active reteplase quantification showed that ∼70 mg soluble reteplase per liter fermentation broth was obtained with 2.35 × 105 IU/mg thrombolytic activity. Finally, fluorescence spectra indicated that the structural conformation of soluble reteplase was identical to its native state. The soluble expression of recombinant reteplase in E. coli was accomplished by co-expression with DsbA/DsbC, which contributes to further research in clinical application and folding mechanism, and provides guidance for production of other proteins with disulfide bonds.

Preparation of ibuprofen-loaded chitosan films for oral mucosal drug delivery using supercritical solution impregnation.

Drug-loaded chitosan films suitable for oral mucosal drug delivery were prepared using supercritical solution impregnation (SSI) technology. Firstly, chitosan films were obtained via casting method, and the film properties including water-uptake, erosion and mucoadhesive were characterized. SSI process was then employed to load the drug of ibuprofen onto the prepared chitosan films, and the effects of impregnation pressure and temperature on morphologies of the ibuprofen-loaded chitosan films and drug loading capacity (DLC) were studied. The SEM and X-ray diffraction patterns suggested that distinct ibuprofen shapes such as microparticles, flake, rod-like and needle-like occurred after impregnation at different pressures, and DLC varied from 7.9% to 130.4% during the SSI process. The ex vivo release profiles showed that ibuprofen-loaded chitosan films could deliver the drug across the rabbit buccal mucosa, and up to 70% of the ibuprofen was released from the matrix in 460 min. SSI process is a promising method to prepare drug-loaded film formulations for oral mucosal drug delivery, which provides the advantages of low solvent residual and sustained- and controlled- release behavior.

Microbial side-chain cleavage of phytosterols by mycobacteria in vegetable oil/aqueous two-phase system.

Microbial side-chain cleavage of natural sterols to 4-androstene-3,17-dione (AD) and 1,4-androstadiene-3,17-dione (ADD) by Mycobacteria has received much attention in pharmaceutical industry, while low yield of the reaction owing to the strong hydrophobicity of sterols is a tough problem to be solved urgently. Eight kinds of vegetable oils, i.e., sunflower, peanut, corn, olive, linseed, walnut, grape seed, and rice oil, were used to construct oil/aqueous biphasic systems in the biotransformation of phytosterols by Mycobacterium sp. MB 3683 cells. The results indicated that vegetable oils are suitable for phytosterol biotransformation. Specially, the yield of AD carried out in sunflower biphasic system (phase ratio of 1:9, oil to aqueous) was greatly increased to 84.8 % with 10 g/L feeding concentration after 120-h transformation at 30 °C and 200 rpm. Distribution coefficients of AD in different oil/aqueous systems were also determined. Because vegetable oils are of low cost and because of their eco-friendly characters, there is a great potential for the application of oil/aqueous two-phase systems in bacteria whole cell biocatalysis.

Supercritical fluid assisted production of chitosan oligomers micrometric powders.

Chitosan oligomers (O-chitosan) micrometric particles were produced from aqueous solution using a novel process, i.e. supercritical fluid assisted atomization introduced by hydrodynamic cavitation mixer (SAA-HCM). Hydrodynamic cavitation was introduced to enhance mass transfer and facilitate the mixing between SC-CO2 and liquid solution for fine particles formation. Well defined, separated and spherical microparticles were obtained, and the particles size could be well controlled with narrow distribution ranging from 0.5 µm to 3 µm. XRD patterns showed amorphous structure of O-chitosan microparticles. FTIR, TGA and DSC analyses confirmed that no change in molecular structure and thermal stability after SAA-HCM processing, while the water content was between 5.8% and 8.4%. Finally, tap densities were determined to be below 0.45 g/cm(3) indicating hollow or porous structures of microparticles. By tuning process parameters, theoretical mass median aerodynamic sizes lied inside respirable range of 1-2 µm, which presented the potential of the O-chitosan microparticles in application as inhaled dry powders. SAA-HCM was demonstrated to be very useful in particle size engineering.

Poly(hydroxyethyl methacrylate)-based composite cryogel with embedded macroporous cellulose beads for the separation of human serum immunoglobulin and albumin.

A novel super-macroporous monolithic composite cryogel was prepared by embedding macroporous cellulose beads into poly(hydroxyethyl methacrylate) cryogel. The cellulose beads were fabricated by using a microchannel liquid-flow focusing and cryopolymerization method, while the composite cryogel was prepared by cryogenic radical polymerization of the hydroxyethyl methacrylate monomer with poly(ethylene glycol) diacrylate as cross-linker together with the cellulose beads. After graft polymerization with (vinylbenzyl)trimethylammonium chloride, the composite cryogel was applied to separate immunoglobulin-G and albumin from human serum. Immunoglobulin-G with a mean purity of 83.2% and albumin with a purity of 98% were obtained, indicating the composite cryogel as a promising chromatographic medium in bioseparation for the isolation of important bioactive proteins like immunoglobulins and albumins.

Bioactive insulin microparticles produced by supercritical fluid assisted atomization with an enhanced mixer.

Supercritical fluid assisted atomization introduced by a hydrodynamic cavitation mixer (SAA-HCM) was used to micronize insulin from aqueous solution without use of any organic solvents. Insulin microparticles produced under different operating conditions including solution type, solution concentration and precipitator temperature presented distinct morphologies such as highly folded, partly deflated, corrugated or smooth hollow spherical shape. Solution concentration had a striking influence on particle size, and insulin microparticles produced from acidic solution had mean diameters increasing from 1.4 µm to 2.7 µm when protein concentration increased from 3g/L to 50 g/L. HPLC chromatograms showed no degradation of insulin after SAA-HCM processing and FTIR, CD and fluorescence data further confirmed the structural stability. TGA analysis revealed that insulin microparticles remained moderate moisture content compared with raw material. In vivo study showed that insulin processed by SAA-HCM from acidic solution retained identical bioactivity. SAA-HCM is demonstrated to be a very promising process for insulin inhaled formulation development.

Preparative enantioseparation of ß-blocker drugs by counter-current chromatography using dialkyl L-tartrate as chiral selector based on borate coordination complex.

Counter-current chromatography (CCC) was applied for preparative enantioseparation of three ß-blocker drugs, including propranolol, pindolol and alprenolol. The two-phase solvent system was composed of chloroform-0.05 mol L(-1) acetate buffer containing 0.10 mol L(-1) boric acid (1:1, v/v), in which 0.10 mol L(-1) di-n-hexyl L-tartrate was added in the organic phase as chiral selector. Influence factors in the enantioseparation of propranolol were investigated. The chromatographic retention mechanism based on borate coordination complex was proposed. 116 mg of racemic propranolol was completely enantioseparated using conventional high speed CCC in a single run, yielding 48 mg of (+)-propranolol with HPLC purity of 98.9% and 47 mg of (-)-propranolol with HPLC purity of 96.3%. Recovery for propranolol enantiomers from CCC fractions was in the range of 75-82%. pH-zone-refining CCC was also successfully applied in enantioseparation of propanolol and it was found that 356 mg of racemic propranolol could be completely enantioseparated. 145 mg of (+)-enantiomer with HPLC purity of 95.6% and 148 mg of (-)-enantiomer with HPLC purity of 98.2% were recovered from pH-zone-refining mode. Separation mechanism about chiral separation by pH-zone-refining CCC was discussed.

Microchannel liquid-flow focusing and cryo-polymerization preparation of supermacroporous cryogel beads for bioseparation.

Polymeric cryogels are sponge-like materials with supermacroporous structure, allowing them to be of interest as new chromatographic supports, cell scaffolds and drug carriers in biological and biomedical areas. The matrices of cryogels are always prepared in the form of monoliths by cryo-polymerization under frozen conditions. However, there are limited investigations on the production of cryogels in the form of adsorbent beads suitable for bioseparation. In this work, we provide a new approach by combining the microchannel liquid-flow focusing with cryo-polymerization for the preparation of polyacrylamide-based supermacroporous cryogel beads with a narrow particle size distribution. The present method was achieved by introducing the aqueous phase solution containing monomer, cross-linker and redox initiators, and the water-immiscible organic oil phase containing surfactant simultaneously into a microchannel with a cross-shaped junction, where the aqueous drops with uniform sizes were generated by the liquid shearing and the segmentation due to the steady flow focusing of the immiscible phase streams. These liquid drops were in situ suspended into the freezing bulk oil phase for cryo-polymerization and the cryogel matrix beads were obtained by thawing after the achievement of polymerization. By grafting the polymer chains containing sulfo binding groups onto these matrix beads, the cation-exchange cryogel beads for protein separation were produced. The results showed that at the aqueous phase velocities from 0.5 to 2.0 cm/s and the total velocities of the water-immiscible phase from 2.0 to 6.0 cm/s, the obtained cryogel beads by the present method have narrow size distributions with most of the bead diameters in the range from 800 to 1500 µm with supermacropores in sizes of about 3-50 µm. These beads also have high porosities with the averaged maximum porosity of 96.9% and the mean effective porosity of 86.2%, which are close to those of the polyacrylamide-based cryogel monoliths. The packed bed using the cryogel beads with mean diameter of 1248 µm, as an example, has reasonable and acceptable liquid dispersion, but high water permeability (4.29 × 10⁻¹° m²) and high bed voidage (90.2%) owing to the supermacropores within the beads, enhanced the rapid binding and separation of protein from the feedstock even at high flow velocities. The purity of the obtained lysozyme from chicken egg white by one-step chromatography using the packed bed was in the range of about 78-92% at the flow velocities of 0.5-15 cm/min, indicating that the present cryogel beads could be an effective chromatographic adsorbent for primary bioseparation.

Optimization of a natural medium for cellulase by a marine Aspergillus niger using response surface methodology.

The components of a natural medium were optimized to produce cellulase from a marine Aspergillus niger under solid state fermentation conditions by response surface methodology. Eichhornia crassipes and natural seawater were used as a major substrate and a source of mineral salts, respectively. Mineral salts of natural seawater could increase cellulase production. Raw corn cob and raw rice straw showed a significant positive effect on cellulase production. The optimum natural medium consisted of 76.9 % E. crassipes (w/w), 8.9 % raw corn cob (w/w), 3.5 % raw rice straw (w/w), 10.7 % raw wheat bran (w/w), and natural seawater (2.33 times the weight of the dry substrates). Incubation for 96 h in the natural medium increased the biomass to the maximum. The cellulase production was 17.80 U/g the dry weight of substrates after incubation for 144 h. The natural medium avoided supplying chemicals and pretreating substrates. It is promising for future practical fermentation of environment-friendly producing cellulase.

Coexpression of chaperonin GroEL/GroES markedly enhanced soluble and functional expression of recombinant human interferon-gamma in Escherichia coli.

Recombinant human interferon-gamma (rhIFN-γ) is a protein of great potential for clinical therapy due to its multiple biological activities. However, overexpressing rhIFN-γ in Escherichia coli was found to accumulate as cytoplasmic inclusion bodies. In this work, a system for soluble and active expression of rhIFN-γ was constructed by coexpressing chaperonin GroEL/GroES in E. coli. The rhIFN-γ gene was fused to a pET-28a expression vector, and rhIFN-γ was partially expressed as the soluble form following coexpression with a second vector producing chaperonin GroEL/GroES. The fermentation of recombinant E. coli harboring rhIFN-γ and GroEL/GroES plasmids was investigated, and the optimized conditions were as follows: culture temperature of 25°C, incubation time of 8 h, isopropyl-ß-D-thio-galactoside concentration of 0.2 mM, and L-arabinose concentration of 0.5 g/L. As a result, the expression level of rhIFN-γ was improved accordingly by 2.2-fold than the control, while a significantly positive correlation was also found between the ratio of supernatant to precipitate of rhIFN-γ and the amount of chaperonin. Circular dichroism spectra, fluorescence spectra, size exclusion chromatography, and chemical crosslinking method were applied to characterize rhIFN-γ, indicating that the three-dimensional structure of rhIFN-γ was identical to that of the native rhIFN-γ. The enzyme-linked immunosorbent assay for active rhIFN-γ quantification showed that coexpression yielded 72.91 mg rhIFN-γ per liter fermentation broth. Finally, protein-protein interactions between rhIFN-γ and chaperonin were analyzed using the yeast two-hybrid system, which provided the direct evidence that chaperonin GroEL/GroES interacted with rhIFN-γ to increase the soluble expression and presented the potential in producing efficiently recombinant proteins.