Dye Affinity Research Articles

Affinity chromatographic separation of secreted alkaline phosphatase and glucoamylase using reactive dyes

A single-step dye affinity chromatographic separation method was developed to separate secreted alkaline phosphatase (SEAP) and glucoamylase produced in CHO cell culture and Aspergillus niger fermentation, respectively. The reactive dye, Procion ® Green H-E4BD, was found to have a good binding capacity for SEAP, whereas Procion ® Blue H-ERD was the best dye ligand for glucoamylase. However, these dyes have a relatively low selectivity for the target protein. Consequently, elution of the adsorbed proteins by KCl solution resulted in a product with many impurity proteins as evident by the multiple protein bands on SDS-PAGE. However, elution of SEAP by its substrate, phosphate, produced a relatively pure protein with a high specific enzyme activity because of the competition for active site between the substrate and the dye ligand. Also, a high-purity glucoamylase product was obtained by elution with a borate solution. The relatively inexpensive dye affinity chromatography thus can be used for purifying enzymes from cell culture and fermentation broths. The adsorption of SEAP on the dye-ligand affinity resin followed the Langmuir isotherm. An axial dispersion model with external mass transfer limitation was developed to simulate the breakthrough curve in the chromatographic column. This mathematical model can be used to scale up the protein adsorption process.

Production and separation of formate dehydrogenase from Candida boidinii

The production of FDH in a 40-l bioreactor was carried out with methanol as the inducer to reach the final intracellular FDH activity of 35 U/g cell. Among different permeabilization methods studied, treatment with toluene at a relatively small amount resulted in the cells with the highest FDH activity. Crude FDH cell extract obtained by ultrasonically breaking down the cell wall was treated with polyethyleneimine (PEI) to separate FDH from other proteins. By adding PEI at a low concentration of 0.04 mg/ml to the cell extract, ∼50% of the proteins formed aggregates with PEI and precipitated; however, FDH was not in these aggregates and remained in the solution. After the PEI treatment, the specific FDH activity increased by 1.6-fold. SDS-PAGE analysis showed that PEI precipitation removed some impurity protein molecules that cannot be separated by affinity chromatography with Sepharose-Procion Blue HERB as the separation ligand, and thus improved the separation efficiency. The adsorbed FDH in the affinity column was eluted with KCl solution. Adding 5 mM NAD + in 0.2 M KCl improved the FDH elution and increased the specific FDH activity by 1.38-fold as compared to elution with 1 M KCl. Overall, the PEI precipitation and dye affinity chromatographic process obtained a high recovery yield of 56% with a 5.5-fold increase in the specific FDH activity from the crude cell extract.

Purification and kinetics of a thermostable laccase from Pycnoporus sanguineus (SCC 108)

Pycnoporous sanguineus was identified as a laccase producer when grown on diluted molasses. The single laccase was purified with a purification factor of 967 by ammonium sulphate precipitation, ion exchange and dye affinity chromatography, to a specific activity of 32.9 U/mg. The molecular mass of 58,000 Da, the optimum pH range between 3 and 5 with ABTS, DMP and guaiacol as substrates and the isoelectric point of 6.7, was similar to some other laccases of filamentous fungi. The optimum temperature was 55 °C and the enzyme displayed enhanced thermal stability with a half-life of 170 min at 75 °C. In terms of k cat/ K m values for ABTS, syringaldazine and DMP, DMP was the best substrate. Kinetic analysis of fifteen more compounds revealed that, the enzyme was a true laccase with a requirement for a free –OH group with an adjacent free or derivatised –OH. The o-diphenols were preferred above their p-counterparts. Compounds with three adjacent –OH groups displayed higher binding affinities but phloroglucinol, an m-substituted phenol was unreactive. The apparent higher stability of this laccase makes it a good candidate for further investigation into it possible application in biotechnology.

Porous dye affinity beads for nickel adsorption from aqueous solutions: A kinetic study

AbstractWe investigated a new adsorbent system, Reactive Red 120 attached poly(2‐hydroxyethyl methacrylate ethylene dimethacrylate) [poly(HEMA–EDMA)] beads, for the removal of Ni2+ ions from aqueous solutions. Poly(HEMA–EDMA) beads were prepared by the modified suspension copolymerization of 2‐hydroxyethyl methacrylate and ethylene dimethacrylate. Reactive Red 120 molecules were covalently attached to the beads. The beads (150–250 μm), having a swelling ratio of 55% and carrying 25.5 μmol of Reactive Red 120/g of polymer, were used in the removal of Ni2+ ions. The adsorption rate and capacity of the Reactive Red 120 attached poly(HEMA–EDMA) beads for Ni2+ ions was investigated in aqueous media containing different amounts of Ni2+ ions (5–35 mg/L) and having different pH values (2.0–7.0). Very high adsorption rates were observed at the beginning, and adsorption equilibria were then gradually achieved in about 60 min. The maximum adsorption of Ni2+ ions onto the Reactive Red 120 attached poly(HEMA–EDMA) beads was 2.83 mg/g at pH 6.0. The nonspecific adsorption of Ni2+ ions onto poly(HEMA–EDMA) beads was negligible (0.1 mg/g). The desorption of Ni2+ ions was studied with 0.1M HNO3. High desorption ratios (>90%) were achieved. The intraparticle diffusion rate constants at various temperatures were calculated as k20°C = 0.565 mg/g min0.5, k30°C = 0.560 mg/g min0.5, and k40°C = 0.385 mg/g min0.5. Adsorption–desorption cycles showed the feasibility of repeated use of this novel adsorbent system. The equilibrium data fitted very well both Langmuir and Freundlich adsorption models. The pseudo‐first‐order kinetic model was used to describe the kinetic data. © 2006 Wiley Periodicals, Inc. J Appl Polym Sci 100:5056–5065, 2006

Molecular cloning and characterization of a plant α1,3/4-fucosidase based on sequence tags from almond fucosidase I

Our work with almond peptide N-glycosidase A made us interested also in the α1,3/4-fucosidase which is used as a specific reagent for glycoconjugate analysis. The enzyme was purified to presumed homogeneity by a series of chromatographic steps including dye affinity and fast-performance anion exchange chromatography. The 63 kDa band was analyzed by tandem mass spectrometry which yielded several partial sequences. A homology search retrieved the hypothetical protein Q8GW72 from Arabidopsis thaliana. This protein has recently been described as being specific for α1,2-linkages. However, cDNA cloning and expression in Pichia pastoris of the A. thaliana fucosidase showed that it hydrolyzed fucose in 3- and 4-linkage to GlcNAc in Lewis determinants whereas neither 2-linked fucose nor fucose in 3-linkage to the innermost GlcNAc residue were attacked. This first cloning of a plant α1,3/4-fucosidase also confirmed the identity of the purified almond enzyme and thus settles the notorious uncertainty about its molecular mass. The α1,3/4-fucosidase from Arabidopsis exhibited striking sequence similarity with an enzyme of similar substrate specificity from Streptomyces sp. (Q9Z4I9) and with putative proteins from rice.

Effect of Ca+ on the photobactericidal efficacy of methylene blue and toluidine blue against gram-negative bacteria and the dye affinity for lipopolysaccharides

Methylene blue (MB) and toluidine blue (TB) form metachromatic complexes with lipopolysaccharides (LPS). The greater photobactericidal efficacy of TB may be explained by its affinity for LPS. This study aims to elucidate the difference in photobactericidal efficacies between the dyes using Ca(2+) as a competitor for dye-binding sites on the bacterial outer membrane. Fixed dye concentration solutions with gram-negative bacteria and increasing concentrations of CaCl(2) were exposed to red laser light. Bacterial survival and spectrophotometry were used to describe the effect of Ca(2+) on dye interaction with bacteria and LPS. MB-mediated photokilling was inhibited more significantly than that of TB. CaCl(2) inhibited dye photobleaching and suppressed the metachromatic reaction between the dyes and LPS, in particular TB. CaCl(2) inhibits bacterial photokilling by binding with LPS, as well as other anionic polymers including outer membrane proteins. LPS is chiefly involved in TB-mediated photokilling, whereas outer membrane proteins probably are more involved in MB-mediated photokilling.

Metabolism of trans, trans-muconaldehyde, a cytotoxic metabolite of benzene, in mouse liver by alcohol dehydrogenase Adh1 and aldehyde reductase AKR1A4

The reductive metabolism of trans, trans-muconaldehyde, a cytotoxic metabolite of benzene, was studied in mouse liver. Using an HPLC-based stopped assay, the primary reduced metabolite was identified as 6-hydroxy-trans, trans-2,4-hexadienal (OH/CHO) and the secondary metabolite as 1,6-dihydroxy-trans, trans-2,4-hexadiene (OH/OH). The main enzymes responsible for the highest levels of reductase activity towards trans, trans-muconaldehyde were purified from mouse liver soluble fraction first by Q-sepharose chromatography followed by either blue or red dye affinity chromatography. In mouse liver, trans, trans-muconaldehyde is predominantly reduced by an NADH-dependent enzyme, which was identified as alcohol dehydrogenase (Adh1). Kinetic constants obtained for trans, trans-muconaldehyde with the native Adh1 enzyme showed a Vmax of 2141+/-500 nmol/min/mg and a Km of 11+/-4 microM. This enzyme was inhibited by pyrazole with a KI of 3.1+/-0.57 microM. Other fractions were found to contain muconaldehyde reductase activity independent of Adh1, and one enzyme was identified as the NADPH-dependent aldehyde reductase AKR1A4. This showed a Vmax of 115 nmol/min/mg and a Km of 15+/-2 microM and was not inhibited by pyrazole.

Chitosan microspheres as immobilized dye affinity support for catalase adsorption

Chitosan microsphere (CS) was prepared by phase-inversion method as the support matrices. Cibacron Blue F3GA (CB) was covalently attached to the chitosan microspheres, and thus the novel dye-affinity adsorbent was obtained. These Cibacron Blue F3GA-attached chitosan microspheres (CB-CS) were used in the catalase (CAT) adsorption studies. The maximum CAT adsorption capacity of Cibacron Blue F3GA-attached chitosan microspheres was 28.4 mg/g at pH 7.0. Langmuir adsorption model was found to be applicable in interpreting CAT adsorption. Significant amount of the adsorbed CAT (up to 90.6%) was eluted in the elution medium containing 0.5 M NaSCN at pH 8.0. It appears that CB-CS can be applied for adsorption of CAT without causing any denaturation.

Magnetic Dye Affinity Beads for the Adsorption of β‐Casein

Casein is well known as a good protein emulsifier and beta-casein is the major component of casein and commercial sodium caseinate. Dye affinity adsorption is increasingly used for protein separation. beta-Casein adsorption onto Reactive Red 120 attached magnetic poly(2-hydroxyethyl methacrylate) (m-PHEMA) beads was investigated in this work. m-PHEMA beads (80-120 microm in diameter) were produced by dispersion polymerization. The dichlorotriazine dye Reactive Red 120 was attached covalently as a ligand. The dye attached beads, having a swelling ratio of 55% (w/w) and carrying different amounts of Reactive Red 120 (9.2 micromol . g(-1)-39.8 micromol . g(-1)), were used in beta-casein adsorption studies. The effects of the initial concentration, pH, ionic strength and temperature on the adsorption efficiency of dye attached beads were studied in a batch reactor. The non-specific adsorption on the m-PHEMA beads was 1.4 mg . g(-1). Reactive Red 120 attachment significantly increased the beta-casein adsorption up to 37.3 mg . g(-1). More than 95.4% of the adsorbed beta-casein was desorbed in 1 h in a desorption medium containing 1.0 M KSCN at pH 8.0. We concluded that Reactive Red 120 attached m-PHEMA beads can be applied for beta-casein adsorption without significant losses in the adsorption capacities.

Nanoclay and Modified Nanoclay as Sorbents for Anionic, Cationic and Nonionic Dyes

The nanoclay, montmorillonite, and some modified nanoclays were used as sorbents for nonionic, anionic and cationic dyes. From the sorption differences among the different dye and clay structures, both chemical and morphological, the sorption forces that played important roles were identified. The nanoclay could easily have a sorption capacity of more than 600 mg sorbate per gram of sorbent at a liquor-to-sorbent ratio of 100 to 1. Furthermore, it could have a sorption of 90% at an initial dye concentration of 6 g/L, or 60% based on the weight of the sorbent, indicating an extremely high dye affinity. This study showed that with certain modifications the nanoclay, montmorillonite, could easily become an excellent sorbent for anionic, cationic and nonionic dyes.

Electrospun cellulose nanofiber as affinity membrane

The objective of this work is to study the feasibility of applying cellulose nanofiber membrane prepared by electrospinning as affinity membrane. Cellulose acetate (CA) solution (0.16 g/ml) in a mixture solvent of acetone/DMF/trifluoroethylene (3:1:1) was electrospun into nonwoven fiber mesh with the fiber diameter ranging from 200 nm to 1 μm. The CA nanofiber mesh was heat treated under 208 °C for 1 h to improve structural integrity and mechanical strength, and then treated in 0.1 M NaOH solution in H 2O/ethanol (4:1) for 24 h to obtain regenerated cellulose (RC) nanofiber mesh, which was used as a novel filtration membrane in this work. The RC nanofiber membrane was further surface functionalized with Cibacron Blue F3GA (CB), a general affinity dye ligand for separation of many biomolecules. The material's chemical and physical properties were studied by SEM, DSC and ATR-FTIR. Water filtration properties of the novel RC membrane were studied and compared with a commercial micro-filtration membrane. The CB derived RC nanofiber membrane has a CB content of 130 μmol/g, and capture capacity of 13 mg/g for bovine serum albumin (BSA) and 4 mg/g for bilirubin. The membrane showed reusability after regeneration with elution buffer. Dynamic adsorption of BSA on the nanofiber membrane was studied by breakthrough curve measurements.

Optical Properties and Morphological Structure of Electron Beam Irradiated Low-Density Polyethylene/Polypropylene Blends

ABSTRACT Blending is an effective method for improving the physical and chemical properties of homopolymers. However, the problem of phase separation often in some polymer blends occurs due to incompatibility of homopolymer, which impairs the expected physico-chemical properties of blends. In this study, isotactic polypropylene (PP) was blended with low density polyethylene (LDPE) in the presence of various ratios of polyfunctional monomer (PFM), namely, trimethylol propane trimethacrylate (TMPTMA) as a cross-linking and/or compatibilizing agents. The electron beam irradiation of the investigated LDPE/PP blends was carried out at different irradiation doses up to 50 kGy to improve the compatibility of LDPE/PP blends. The dye affinity for disperse and basic dyes of LDPE, PP, and LDPE/PP blends was investigated in terms of color strength and visible absorbance intensities. In addition, the microstructures of the investigated samples have been studied, and it is considered as an indication of the compatibility of LDPE/PP blend. The results show that the unmodified and PFM modified LDPE samples show the highest values of both color strength and visible absorbance intensities compared with unmodified and PFM modified PP samples. The intensities of these values in the LDPE/PP blends also decrease as the PP content increases in the blend. In addition, the increase of EB irradiation dose and PFM ratios leads to an increase in the values of color strength and visible absorbance intensities. Moreover, the affinity of all investigated samples toward disperse dye is being more pronounced than the basic dye. The interfacial adhesion of LDPE/PP blend could be enhanced by adding cross-linking agent (TMPTMA) and EB irradiation process with different irradiation doses. The obtained data for the microstructures of all the investigated samples support that the EB irradiation beside the incorporation of PFM into the homopolymer matrix remarkably improve the compatibility of the polymer blend.

Bilirubin removal from human plasma by Cibacron Blue F3GA using immobilized microporous affinity membranous capillary method

A novel affinity sorbent system for direct bilirubin removal from human plasma was developed. These new adsorbents comprise Cibacron Blue F3GA as the specific ligand, and microporous membranous poly(tetrafluoroethylene) capillary (modified by coating with a hydrophilic layer of poly(vinyl alcohol) after activation) as the carrier matrix. The affinity adsorbents carrying 126.5 μmol Cibacron Blue F3GA/g polymer was then used to remove bilirubin in a flow-injection system. Non-specific adsorption on the poly(vinyl alcohol) coated capillary remains low, and higher affinity adsorption capacity, of up to 76.2 mg/g polymer was obtained after dye immobilization. The bilirubin adsorption capacity of the affinity capillary decreased with increase in the recirculation rate of plasma. The adsorption capacity increased with increase the temperature while decreased with increase the ionic strength. The maximum adsorption was only observed in neutral solution (pH 6–7). The adsorption isotherm fitted the Langmuir model well. These new adsorbents have higher velocity of mass transfer, better adsorption capacity, less fouling, longer service life and good reusability. The results of blood tests suggested the dye affinity capillary has good blood compatibility.

Poly(Styrene‐Hydroxyethyl Methacrylate) Monodisperse Microspheres as Specific Sorbent in Dye Affinity Adsorption of Albumin

In this study, human serum albumin (HSA) adsorption properties of reactive green HE‐4BD‐attached monodisperse poly(styrene‐2‐hydroxyethyl methacrylate) [poly(St‐HEMA)] microspheres were investigated. Poly(St‐HEMA) microspheres with an uniform size of 4.0 µm in diameter were produced by the dispersion copolymerization of St and HEMA in an ethanol–water medium. Reactive green HE‐4BD was covalently attached onto the poly(St‐HEMA) microspheres via a nucleophilic substitution reaction between the hydroxyl groups of HEMA and triazinyl chloro groups of dye with the equilibrium coupling capacity of 45.8 µmol dye (g polymer)−1. The poly(St‐HEMA) monodisperse microspheres were characterized by scanning electron microscopy. The effect of concentration of HSA, medium pH, and ionic strength on the adsorption efficiency of dye‐attached microspheres were studied in a batch system. The nonspecific adsorption of HSA on the poly(St‐HEMA) microspheres was 2.1 mg g−1. Reactive green HE‐4BD attachment significantly increased the HSA adsorption up to 221 mg g−1. The langmuir adsorption model was found to be applicable in interpreting HSA adsorption by reactive green HE‐4BD attached microspheres. Significant amount of the adsorbed HSA (up to 92%) was eluted in 1 hr in the elution medium containing 1.0‐M NaCl. To determine the effects of adsorption conditions on possible conformational changes of HSA structure, fluorescence spectrophotometry was employed. We resulted that poly(St‐HEMA) dye‐affinity microspheres can be applied for HSA adsorption without causing any significant conformational changes. Repeated adsorption/elution processes showed that these dye‐attached monodisperse microspheres are suitable for HSA adsorption.

An Integrated Process for Separation of Major and Minor Proteins From Goat Serum

Analysis of minor proteins in animal sera is of considerable clinical significance. To be able to detect these proteins, depletion of major proteins (albumin and immunoglobulin G [IgG]) is necessary. Many of these proteins are also required in pure form for a variety of biochemical applications. The present work uses goat serum as the system and describes the separation and purification of both major and several minor proteins. This was carried out by judicious adaptation and combination of separation technologies such as immobilized metal ion affinity chromatography (on a somewhat novel matrix), dye affinity chromatography, and lectin affinity chromatography. Albumin, IgG, alpha2-macroglobulin, alpha1-proteinase inhibitor, and transferrin were obtained from the serum. The purified preparations were found to be homogeneous on sodium dodecyl sulfate polyacrylamide gel electrophoresis.

Surface Modification and Characterization of Magnetic Polymer Nanospheres Prepared by Miniemulsion Polymerization

A novel and effective protocol for the surface modification and quantitative characterization of magnetic polymeric nanospheres prepared by miniemulsion polymerization is reported. Composite nanospheres consisting of polymer-coated iron oxide nanoparticles were prepared by the miniemulsion polymerization of methyl methacrylate and divinylbenzene in the presence of magnetic fluid. Surface modification reaction of the magnetic polymer with poly(ethylene glycol) (PEG) was employed to obtain a hydrophilic hydroxyl-group-functionalized magnetic nanospheres. An affinity dye, Cibacron blue F3G-A (CB), was then coupled covalently to prepare a magnetic nonporous affinity adsorbent. The morphology and magnetic property of the polymer nanospheres obtained were examined by transmission electron microscopy and a vibrating sample magnetometer. The contents of surface groups modified were quantitatively measured by using diffusive reflectance Fourier transform infrared spectroscopy on the basis of a linear relationship between the intensity ratio of IC-O-C/IC=O and the content of PEG. X-ray photoelectron spectroscopy (XPS) was used to examine the surface of magnetic nanospheres. It was confirmed by the comparison of XPS spectra of both dye-coated and uncoated magnetic nanospheres to which the CB ligand was coupled, and the surface of the PEG-modified nanospheres had an exact 3:7 atomic ratio of sulfur to nitrogen.

The acrosomal vesicle of mouse sperm is a calcium store.

Subsequent to binding to the zona pellucida, mammalian sperm undergo a regulated sequence of events that ultimately lead to acrosomal exocytosis. Like most regulated exocytotic processes, a rise in intracellular calcium is sufficient to trigger this event although the precise mechanism of how this is achieved is still unclear. Numerous studies on mouse sperm have indicated that a voltage-operated Ca2+ channel plays some immediate role following sperm binding to the zona pellucida glycoprotein ZP3. However, there is also evidence that the mammalian sperm acrosome contains a high density of IP3 receptors, suggesting that the exocytotic event involves the release of Ca2+ from the acrosome. The release of Ca2+ from the acrosome may directly trigger exocytosis or may activate store-operated Ca2+ channels in the plasma membrane. To test the hypothesis that the acrosome is an intracellular store we loaded mammalian sperm with the membrane permeant forms of three Ca2+-sensitive fluorescent indicator dyes: fura-2, indo-1, and Calcium Green-5N. Fluorescence microscopy revealed that the sperm were labeled in all intracellular compartments. When fura-2 labeled sperm were treated with 150 microM MnCl2 to quench all fluorescence in the cytosol, or when the sperm were labeled with the low affinity dye Calcium Green-5N, there was a large Ca2+ signal in the acrosome. Consistent with the acrosome serving as an intracellular Ca2+ reservoir, the addition of 20 microM thapsigargin, a potent inhibitor of the smooth endoplasmic reticular Ca2+-ATPase (SERCA), to populations of capacitated sperm resulted in nearly 100% acrosomal exocytosis within 60 min (tau1/2 approximately 10 min), in the absence of extracellular Ca2+. Additionally, treatment of sperm with 100 microM thimerosal, an IP3 receptor agonist, also resulted in acrosomal exocytosis. Taken together, these data suggest that the mouse sperm acrosome is a Ca2+ store that regulates its own exocytosis through an IP3 Ca2+ mobilization pathway.

Dye affinity hollow fibers for albumin purification.

Reactive Green HE 4BD was immobilized on polyamide (PA) hollow fibers for human serum albumin (HSA) adsorption from both aqueous solutions and human plasma. Different amounts of Reactive Green HE 4BD were incorporated on the PA hollow fibers by changing the dye attachment conditions, i.e. the initial dye concentration and the addition of sodium carbonate and sodium chloride. The maximum amount of dye attachment was obtained as 39.4 micromol x g(-1) when the hollow fibers were treated with 3 M HCl for 30 min before performing the dye attachment. HSA adsorption onto unmodified and dye-attached hollow fibers was investigated batchwise. The non-specific adsorption of HSA was low (6.0 mg/g hollow fiber). Dye attachment onto the hollow fibers significantly increased the HSA adsorption (86.7 mg/g). The maximum HSA adsorption was observed at pH 5.0. Higher HSA adsorption was observed from human plasma (198 mg HSA/g). The desorptions were performed by adding 0.1 M Tris/HCl buffer containing 0.5 M NaSCN or 1.0 M NaCl to the HSA solutions in which adsorption equilibria had been reached. The desorption results demonstrated that the adsorption of HSA to the adsorbent was reversible. Chemical structure of Reactive Green HE-4BD.

Cibacron Blue F3GA‐attached magnetic poly(2‐hydroxyethyl methacrylate) beads for human serum albumin adsorption

AbstractAn affinity dye ligand, Cibacron Blue F3GA, was covalently attached onto magnetic poly(2‐hydroxyethyl methacrylate) (mPHEMA) beads for human serum albumin (HSA) adsorption from both aqueous solutions and human plasma. The mPHEMA beads, in the size range of 80 to 120 µm, were prepared by a modified suspension technique. Cibacron Blue F3GA molecules were incorporated on to the mPHEMA beads. The maximum amount of Cibacron Blue F3GA attachment was obtained as 68.3 µmol g−1. HSA adsorption onto unmodified and Cibacron Blue F3GA‐attached mPHEMA beads was investigated batchwise. The non‐specific adsorption of HSA was very low (1.8 mg g−1). Cibacron Blue F3GA attachment onto the beads significantly increased the HSA adsorption (94.5 mg g−1). The maximum HSA adsorption was observed at pH 5.0. Higher HSA adsorption was observed from human plasma (138.3 mg HSA g−1). Desorption of HSA from Cibacron Blue F3GA‐attached mPHEMA beads was obtained by using 0.1 M Tris/HCl buffer containing 0.5 M NaSCN. High desorption ratios (up to 98% of the adsorbed HSA) were observed. It was possible to re‐use Cibacron Blue F3GA‐attached mPHEMA beads without any significant decreases in their adsorption capacities. Copyright © 2004 Society of Chemical Industry

Human pituitary glutaminyl cyclase: expression in insect cells and dye affinity purification

Human pituitary glutaminyl cyclase (hQC) was expressed in Drosophila S2 cells under the control of an inducible metallothionene promoter and fused to the Drosophila immunoglobulin-binding protein signal sequence to enable secretion into the culture media. Expression levels reached 50 μg/mL culture media after 7 days of induction. The enzyme was purified to homogeneity directly from culture media by affinity chromatography on Reactive Blue 4–agarose using a step pH elution. The identity of the expressed protein was confirmed by peptide mass mapping and Western blotting. Glutaminyl cyclase was expressed as a fully active 37 kDa enzyme with k cat/ K m values of 14.3, 9.3, and 2.4 mM −1 s −1 for the substrates Gln–Gln, Gln–NH 2, and Gln- t-butyl ester, respectively. The two cysteines were disulfide bonded, and the lone predicted glycosylation site, asparagine 49, was shown by both enzymatic deglycosylation of the expressed enzyme and site-directed mutagenesis to be glycosylated.