Selective adsorption of endotoxin inside a polycationic network of flat-sheet microfiltration membranes

For the removal of remaining amounts of endotoxin, sorbents with high selectivity for endotoxin are required. Typically, particulate sorbents with positively charged ligands, such as histidine, polymyxin B poly-L-lysine and poly(ethyleneimine) (PEI), display moderate to high removal efficiencies in an environment of low ionic strength. It was found that polycationic ligands are most suitable to meet an endotoxin concentration which is below the threshold level required for parenteralia. Furthermore, protein recoveries close to 100% are obtained if the decontamination is performed at a pH close to the pI of acidic proteins. The high selectivity is probably caused by complexation of the polycationic ligand with the polyanionic endotoxin, leading to interactions with KD < 10(-9) M using PEI and assuming M(r) = 10 kDa for monomeric endotoxin; with BSA the same ligand reveals only KD = 4 x 10(-6) M. Using polymer-coated microfiltration membranes, immobilization of positively charged ligands leads to membrane adsorbers which are generally superior to chromatographic adsorbers and allow faster processing. Since immobilization takes place at polymer chains, low-molecular-weight ligands mainly add positive charges to the hydrophilic polymer. Consequently, membrane adsorbers with low-molecular-weight ligands, even DEAE, demonstrate similar selectivity to PEI or poly-L-lysine.     

Improvement of Heterologous Protein Production in Aspergillus oryzae by RNA Interference with α-Amylase Genes

Aspergillus oryzae RIB40 has three α-amylase genes (amyA, amyB, and amyC), and secretes α-amylase abundantly. However, large amounts of endogenous secretory proteins such as α-amylase can compete with heterologous protein in the secretory pathway and decrease its production yields. In this study, we examined the effects of suppression of α-amylase on heterologous protein production in A. oryzae, using the bovine chymosin (CHY) as a reporter heterologous protein. The three α-amylase genes in A. oryzae have nearly identical DNA sequences from those promoters to the coding regions. Hence we performed silencing of α-amylase genes by RNA interference (RNAi) in the A. oryzae CHY producing strain. The silenced strains exhibited a reduction in α-amylase activity and an increase in CHY production in the culture medium. This result suggests that suppression of α-amylase is effective in heterologous protein production in A. oryzae.     

Endotoxin removal with poly(ethyleneimine)-immobilized adsorbers: Sepharose 4B versus flat sheet and hollow fibre membranes

Poly(ethyleneimine) was immobilized on poly(vinyl alcohol)-coated nylon flat sheet membranes, poly(vinyl alcohol) and poly(ethylenevinyl alcohol) hollow fibre membranes as well as Sepharose 4B. The resulting poly(ethyleneimine)-immobilized adsorbers were used for removal of E. coli derived endotoxin from buffers and bovine serum albumin solutions. The efficiency of poly(ethyleneimine) proved to be constant over a wide pH range, including phosphate buffered saline. The performance depended upon the matrix type employed: endotoxin clearance factors varied from 100 to 120 000 in protein-free solutions and 40 to 33 000 in solutions of bovine serum albumin using 6000 EU/ml as feed concentration. The best adsorber was the flat sheet membrane-immobilized poly(ethyleneimine), followed by the hollow fibre-immobilized poly(ethyleneimine) and poly(ethyleneimine)-Sepharose. The factors influencing endotoxin clearance were the mass transport (convective systems were superior to the diffusive system), the chemical composition and the surface structure of the underlying matrix.     

Membrane cartridges for endotoxin removal from interferon preparations

The suitability of membrane cartridges for the removal of endotoxin from both distilled water and interferon preparations was examined. The endotoxin concentrations were reduced to 4.0 and 7.3 EU/ml, respectively, when about 4000 ml of distilled water with 20 and 28 EU/ml were passed through the deoxycholate and chitosan immobilized membrane cartridges. When 200 ml of interferon preparation with endotoxin concentration more than 80 EU/ml and pH 3.9 were applied to a deoxycholate immobilized membrane cartridge at a flow-rate of 9 ml/min, the endotoxin concentration was reduced to less than 10 EU/ml. However, if an interferon preparation of 450 ml, with more than 80 EU/ml of endotoxin and pH 3.9 was applied to the chitosan immobilized membrane cartridge at a flow-rate of 18 ml/min, the endotoxin concentration was reduced to less than 10 EU/ml.     

Structural and functional characterization of recombinant medaka fish alpha-amylase expressed in yeast Pichia pastoris

The medaka fish α-amylase was expressed and purified. The expression systems were constructed using methylotrophic yeast Pichia pastoris, and the recombinant proteins were secreted into the culture medium. Purified recombinant α-amylase exhibited starch hydrolysis activity. The optimal pH, denaturation temperature, and K_M and V_max values were determined; chloride ions were essential for enzyme activity. The purified protein was also crystallized and examined by X-ray crystallography. The structure has the (α/β)₈ barrel fold, as do other known α-amylases, and the overall structure is very similar to the structure of vertebrate (human and pig) α-amylases. A novel expression plasmid was developed. Using this plasmid, high-throughput construction of an expression system by homologous recombination in P. pastoris cells, previously reported for membrane proteins, was successfully applied to the secretory protein.     

Optimization of the Endotoxin Removal Performance of Solid-Phase Conjugated S3E3 Antimicrobial Peptide Using Response Surface Methodology

S3E3 is a new variant of S3 antimicrobial peptide (AMP) derived from factor C of horseshoe hemolymph and features a high binding affinity for endotoxin. In this research, site-specific conjugated S3E3 AMP onto Sepharose 6% solid phase support (S3E3-S-Sepharose) was applied for endotoxin removal from protein solutions. The bovine serum albumin (BSA) was chosen as a protein model due to its acidic-sticky nature interfering with the endotoxin removal process. The batch process parameters including, endotoxin concentration, pH, and ionic strength of the sample were optimized by response surface methodology to reach maximum endotoxin binding capacity and protein recovery. The predicted optimal conditions for enhanced endotoxin removal performance were as follows: pH 4.5, 25 mM NaCl, and 68,500 EU/ml endotoxin leading to a maximum endotoxin binding capacity of 3.114?×?10⁺⁶ EU/ml of resin and a 95.89% protein recovery. S3E3-S-Sepharose could be applied as an efficient endotoxin removal affinity chromatography matrix at downstream processes of recombinant therapeutics due to its high capacity and protein recovery.

     

Exploring the binding interactions of structurally diverse dichalcogenoimidodiphosphinate ligands with α-amylase: Spectroscopic approach coupled with molecular docking

Postprandial hyperglycemia has orchestrated untimely death among diabetic patients over the decades and regulation of α-amylase activity is now becoming a promising management option for type 2 diabetes. The present study investigated the binding interactions of three structurally diverse dichalcogenoimidodiphosphinate ligands with α-amylase to ascertain the affinity of the ligands for α-amylase using spectroscopic and molecular docking methods. The ligands were characterized using ¹H and ³¹P NMR spectroscopy and CHN analysis. Diselenoimidodiphosphinate ligand (DY300), dithioimidodiphosphinate ligand (DY301), and thioselenoimidodiphosphinate ligand (DY302) quenched the intrinsic fluorescence intensity of α-amylase via a static quenching mechanism with bimolecular quenching constant (Kq) values in the order of x10¹¹ M^-1s^-1, indicating formation of enzyme-ligand complexes. A binding stoichiometry of n≈1 was observed for α-amylase, with high binding constants (Ka). α-Amylase inhibition was as follow: Acarbose > DY301>DY300>DY302. Values of thermodynamic parameters obtained at temperatures investigated (298, 304 and 310 K) revealed spontaneous complex formation (ΔG<0) between the ligands and α-amylase; the main driving forces were hydrophobic interactions (with DY300, DY301, except DY302). UV–visible spectroscopy and Förster resonance energy transfer (FRET) affirmed change in enzyme conformation and binding occurrence. Molecular docking revealed ligands interaction with α-amylase via some key catalytic site amino acid residues (Asp197, Glu233 and Asp300). DY301 perhaps showed highest α-amylase inhibition (IC₅₀, 268.11 ± 0.74 μM) due to its moderately high affinity and composition of two sulphide bonds unlike the others. This study might provide theoretical basis for development of novel α-amylase inhibitors from dichalcogenoimidodiphosphinate ligands for management of postprandial hyperglycemia.     

Control of protein synthesis in barley aleurone layers by the plant hormones gibberellic acid and abscisic acid

The patterns of protein synthesis in barley aleurone layers treated with gibberellic acid (GA₃) and abscisic acid (ABA) are compared with the patterns observed in wheat germ in vitro translation assays directed by RNA isolated from similarly treated layers. When used alone, GA₃ and ABA both induce the formation of new translatable mRNAs and cause new proteins to be synthesized. The effects of GA₃ are more dramatic than those of ABA. In GA₃-treated tissues, overall protein synthesis is redirected to produce large quantities of α-amylase and a few other GA₃-induced proteins, while other protein synthesis is reduced or stopped. Large amounts of new translatable mRNA for α-amylase are also induced such that the dominant in vitro translation product is α-amylase. These changes are blocked by the simultaneous addition of ABA to the tissue. In GA₃ plus ABA-treated layers, few changes in protein synthesis in vivo are observed when compared to protein synthesis in untreated tissue, although the induction of mRNA for α-amylase and the other GA₃-induced mRNAs does occur. This indicates that ABA does not interfere with GA₃ induction of translatable mRNAs but prevents the translation of these mRNAs in vivo. Thus ABA and potentially GA₃ regulate the translation of proteins in vivo in barley aleurone layers.     

Endotoxin removal by affinity sorbents.

Affinity sorbents and detoxification strategies are described to remove different amounts of endotoxin. Advantages and disadvantages of the employed ligands are discussed and it is shown that both electrostatic and hydrophobic interactions contribute to the association of ligands and endotoxins. Furthermore, the flexibility of the ligand is more important than an exact structural match between ligand and ligate. Owing to the formation of endotoxin micelles and vesicles, microfiltration membrane adsorbers are particularly effective since mass transfer restrictions are almost absent in the flow-through pores.     

Factors affecting endotoxin removal from recombinant therapeutic proteins by anion exchange chromatography

Removal of endotoxins from recombinant proteins is a critical and challenging step in the preparation of injectable therapeutics, as endotoxin is a natural component of the bacterial expression systems widely used to manufacture therapeutic proteins. In this study we investigated various parameters affecting anion exchange chromatography to selectively remove endotoxins from therapeutic proteins. NY-ESO-1, Melan-A, and SSX-2 are different recombinant proteins used in this study, all of them are cancer antigens currently developed as potential immunotherapeutic agents. We found that by using a commercially available Q XL resin in a flow-through mode, endotoxin could be effectively removed from these proteins while maintaining very acceptable protein yields. The ratio of resin volume to endotoxin load was analyzed to determine the endotoxin binding capacity of the resin. In our hands at least 900,000 endotoxin units (EU) could be loaded per ml of Q XL resin. Solution conductivity could be increased to 20 mS/cm to minimize protein loss by weakening protein-resin attraction, and pH could be increased to enhance endotoxin removal by weakening endotoxin-protein attraction. Endotoxin levels were ultimately decreased to below 0.5 EU per microg of protein, an over 2000-fold reduction in this single step. A successful scale-up of these processes in which column volume was increased 100-fold was performed under cGMP conditions with over 80% protein recovery.     

High-performance affinity capture-removal of bacterial pyrogen from solutions

Synthetic peptide S3Δ has high affinity for bacterial endotoxin or lipopolysaccharide (LPS). Under tested conditions of pH 5–9 and 0–0.4 M NaCl, the affinity constant, K_D ranged from 2·10⁻⁶ to 2·10⁻⁹ M⁻¹. A novel affinity matrix based on peptide S3Δ was developed for removal of LPS from solutions such as: water; buffers with a wide range of ionic strength and pH; medium for cell culture; and protein solutions under optimized conditions. At a starting LPS of ≈100 EU/ml, a post-purification level below 0.005 EU/ml was achieved.     

Level of bacterial endotoxins in Haemophilus influenzae type b vaccines conjugated with toxoids (td, Di)

Quantitative evaluation of bacterial endotoxin was performed in the following vaccines: Act-Hib, Hiberix, Hib-Titer. The aim of this study was to assess the accuracy and precision of chromogenic LAL test with S-2423 substrate for this particular biopreparations and after that to determine the amounts of endotoxin as a factor of vaccine safety. Because of the lack of information concerning the presence of endotoxin in Act-Hib vaccine, we also tried to establish the limits for the presence of endotoxin in this type of vaccine. The estimated level of endotoxin was as follows: 110 EU/ml in Act-Hib, 1.64 EU/ml in Hiberix and 2.4 EU/ml in Hib-Titer. The results of this study showed that the amounts of endotoxin was dependent on the molecular size of polysaccharide PRP and on the presence of protein component. The limit of endotoxin presence in Act-Hib vaccine recommended by us is max. 150 EU/ml.     

Plasma-mediated enhancement of enzyme secretion in Aspergillus oryzae

Technical bottlenecks in protein production and secretion often limit the efficient and robust industrial use of microbial enzymes. The potential of non-thermal atmospheric pressure plasma to overcome these technical barriers was examined. Spores of the fermenting fungus Aspergillus oryzae (A. oryzae) were submerged in potato dextrose broth (PDB) (5 × 10⁶ per ml) and treated with micro dielectric barrier discharge plasma at an input voltage of 1.2 kV and current of 50 to 63 mA using nitrogen as the feed gas. The specific activity of α-amylase in the broth was increased by 7.4 to 9.3% after 24 and 48 h of plasma treatment. Long-lived species, such as NO₂⁻ and NO₃⁻, generated in PDB after plasma treatment may have contributed to the elevated secretion of α-amylase. Observations after 24 h of plasma treatment also included increased accumulation of vesicles at the hyphal tip, hyphal membrane depolarization and higher intracellular Ca²⁺ levels. These results suggest that long-lived nitrogen species generated in PDB after plasma treatment can enhance the secretion of α-amylase from fungal hyphae by depolarizing the cell membrane and activating Ca²⁺ influx into hyphal cells, eventually leading to the accumulation of secretory vesicles near the hyphal tips.     

New Specific α-Glucosidase Inhibitor Flavonoid from Thymelaea tartonraira Leaves: Structure Elucidation,Biological and Molecular Docking Studies

The phytochemical investigation of Thymelaea tartonraira leaves led to the isolation and characterization of six compounds, including one new flavonoid glycoside identified as hypolaetin 8-O-β-D-galactopyranoside ( 4 ) along with five known compounds, daphnoretin ( 1 ), triumbelletin ( 2 ), genkwanin ( 3 ), tiliroside ( 5 ) and yuankanin ( 6 ). Their structures were established based on spectroscopic methods, such as UV, IR, NMR, and HR-ESI-MS. Triumbelletin ( 2 ) and tiliroside ( 5 ) were isolated for the first time from T. tartonraira leaves. The antioxidant property of all isolated compounds was tested based on DPPH, FRAP and total antioxidant capacity assays. Compound 4 displayed an antioxidant potency more interesting than vitamin C with an IC₅₀=15.00±0.50 μg/ml, followed by compound 5 . Furthermore, the both compounds 4 and 5 were tested for their α-amylase inhibitory activity in-vitro. Compound 4 displayed higher potency to inhibit α-amylase, with an IC₅₀=46.49±2.32 μg/ml, than compound 5 , with an IC₅₀=184.2±9.2 μg/ml, while the reference compound acarbose presented the highest potency to inhibit α-amylase with an IC₅₀=0.44±0.022 μg/ml. Compound 4 displayed a strong inhibitory ability of α-glucosidase activity approximately twice more than the reference compound, acarbose, with IC₅₀ values of 60.00±3.00 and 125.00±6.25 μg/ml, respectively. Thus, compound 4 exhibited a specific inhibitory activity for α-glucosidase. The molecular docking studies have supported our findings and suggested that compound 4 has been involved in various binding interactions within the active site of both enzymes α-amylase and α-glucosidase.     

Strategies for the depyrogenation of contaminated immunoglobulin G solutions by histidine-immobilized hollow fiber membrane

The depyrogenation of different IgG solutions using the histidine-linked hollow fiber membrane developed in our laboratory is presented here. Three strategies for endotoxin (ET) removal were investigated according to the immobilized histidine's ability to bind different immunoglobulins: (1) ET removal from 1 mg/ml non histidine-binding mouse monoclonal IgG₁ (MabCD4) solution was achieved in the presence of acetate buffer (pH 5.0) without any protein loss. (2) For contaminated human IgG, combined adsorption of ET and IgG in the presence of MOPS or Tris buffer was tested, followed by differential elution using increasing salt concentrations. This attempt was not successful since ET were quantitatively found in the IgG elution fraction. (3) Alternatively, it was proposed to adsorb selectively ET in the presence of acetate buffer (pH 5.0) under non binding conditions for human IgG. Human IgG could then be purified if necessary with the same membrane in the presence of MOPS buffer (pH 6.5). With a 1 m² histidine-PEVA module under these operating conditions, it is estimated that the depyrogenation of 3 1 of 1 mg/ml IgG (human or murine) solution containing 80 EU/ml of ET should be possible.     

Membrane mutation related to the production of extracellular -amylase and protease in bacillus subtilis

Mutants which had a genetic character to increase the production of both α-amylase and protease simultaneously, were isolated from a transformable strain of $\text{Bacillus}$$\text{subtilis}$ Marburg by NTG treatment. This mutation seems to have occurred at a single gene of the bacterial chromosome and was not linked to $\text{aro}$₁₁₆ which was closely linked to the α-amylase gene. When this mutation and an α-amylase regulator gene ($\text{amyR}$^h) coexisted in one strain, their synergistic effect on extracellular α-amylase production ws observed. The introduction of this mutation resulted in a loss of competence for the transformation. The SDS disc gel electrophoretic profiles of the membrane proteins from the original strain, the mutants and transformants with this mutation showed a remarkable difference in one component.     

Structural and functional characterization of recombinant medaka fish alpha-amylase expressed in yeast Pichia pastoris

The medaka fish α-amylase was expressed and purified. The expression systems were constructed using methylotrophic yeast Pichia pastoris, and the recombinant proteins were secreted into the culture medium. Purified recombinant α-amylase exhibited starch hydrolysis activity. The optimal pH, denaturation temperature, and K(M) and V(max) values were determined; chloride ions were essential for enzyme activity. The purified protein was also crystallized and examined by X-ray crystallography. The structure has the (α/β)(8) barrel fold, as do other known α-amylases, and the overall structure is very similar to the structure of vertebrate (human and pig) α-amylases. A novel expression plasmid was developed. Using this plasmid, high-throughput construction of an expression system by homologous recombination in P. pastoris cells, previously reported for membrane proteins, was successfully applied to the secretory protein.     

Calcium regulation of the secretion of α-amylase isoenzymes and other proteins from barley aleurone layers

The effect of calcium on the secretion of α-amylase (EC 3.2.1.1) and other hydrolases from aleurone layers of barley (Hordeum vulgare L. cv. Himalaya) was studied. Withdrawal of Ca²⁺ from the incubation medium of aleurone layers preincubated in 5 μM gibberellic acid (GA₃) and 5 mM CaCl₂ results in a 70–80% reduction in the secretion of α-amylase activity to the incubation medium. Agar-gel electrophoresis shows that the reduction in α-amylase activity following Ca²⁺ withdrawal is correlated with the disappearance of group B isoenzymes from the incubation medium. The secretion of isoenzymes of group A is unaffected by Ca²⁺. The addition of Ca²⁺ stimulates the secretion of group-B isoenzymes but has no measurable effect on either the α-amylase activity or the isoenzyme pattern of aleurone-layer extracts. Pulse-labelling experiments with [³⁵S]methionine show that Ca²⁺ withdrawal results in a reduction in the secretion of labelled polypeptides into the incubation medium. Immunochemical studies also show that, in the absence of Ca²⁺, α-amylase isoenzymes of group B are not secreted into the incubation medium. In addition to its effect on α-amylase, Ca²⁺ influences the secretion of other proteins including several acid hydrolases. The secretion of these other proteins shows the same dependence on Ca²⁺ concentration as does that of α-amylase. Other cations can promote the secretion of α-amylase to less and varying extents. Strontium is 85% as effective as Ca²⁺ while Ba²⁺ is only 10% as effective. We conclude that Ca²⁺ regulates the secretion of enzymes and other proteins from the aleurone layer of barley.