New biospecific adsorbents for the purification of estradiol receptor

The synthesis of biospecific adsorbents for the purification of the cytosol estrogen receptor from calf uterus is described. The characteristic of several estradiol derivatives, spacer chains, and insoluble matrix were systematically studied. Estradiol derivatives substituted at positions C2, 3, 4 7 alpha, 17 alpha, and 17 beta were tested for their affinities for the receptor; positions 7 alpha and 17 alpha were the most suibable. Acidic compounds had lower affinities than their methylester analogues. Long chain derivatives bound the receptor less firmly than corresponding shorter chains. However, when these ligands were attached to an insoluble matrix, the long spacer chain derivatives (greater than or equal to 14 atoms) were more efficient than the shorter ones. There was a satisfactory parallelism between affinities of free ligands and receptor binding to the respective biospecific adsorbents. On the basis of their stability in the presence of cytosol (no release of ligand), due to the absence of ester bonds, long chains were selected as spacers. Both acrylamide and agarose biospecific adsorbents displayed some ionic exchange capacity and consequently nonspecifically bound proteins; the influence of this nonspecific binding on the purification of the receptor was studied. On the basis of their stability, of their binding specificity, and of their selectivity for the receptor, the estradiol-7 alpha derivative adsorbents were selected for the purification of the receptor.     

Synthesis, characterization and potential application of monoacyl-cyclodextrins

Although the preparation of cyclodextrin (CD) monoesters with a variety of carboxylic acids has been already described in the literature, the direct regioselective CD acylation has proved to be critical, often requiring to be replaced with a more elaborate synthetic process. In this paper we describe the one-step preparation of several monoacylated CDs from acyclic or aromatic carboxylic acid derivatives. The ability of β-CD to enclose cupric ions in a sandwich-type manner was exploited to lead to high regioselectivity in the acylation of β-CD with benzoyl chloride, cinnamoyl chloride and phenyl acetyl chloride in water. Long chain aliphatic monoesters of α-, β- and γ-CD were best prepared in DMF. The results of our study showed that solvent and general conditions determined an overwhelming regioselectivity of acylation. ¹H, ¹³C and 2D NMR experiments could easily discriminate the position of the ester. Monoacylated CDs were evaluated as a carrier of silibinin, the inclusion complexes were prepared and characterized by thermal analysis.     

Effects of poly(ethylene glycol) and salt on the binding of α-amylase from the fermentation broth of Bacillus amyloliquefaciens by Cu2+-β-CD affinity adsorbent

α-Amylase from Bacillus amyloliquefaciens was purified by the immobilized metal ion affinity adsorbent, β-CD_cl-IDA-Cu²⁺. The adsorbent was prepared by reacting the cross-linked β-cyclodextrin (β-CD) with the ligand, iminodiacetic acid (IDA). The copper ion was further linked to the adsorbent. Poly(ethylene glycol) (PEG) was added to the fermentation broth to improve the adsorption efficiency of the adsorbent toward α-amylase. The effort was to provide hydrophobic interactions with the impurities which might interfere with the adsorption of α-amylase. It also provided a polymer shielding effect to prevent non-specific interactions. With the addition of PEG, the adsorption efficiency could be increased to 98%. Imidazole containing a phosphate buffer and NaCl was used to elute the bound α-amylase. By consecutive adsorption/desorption steps, up to 81% of the α-amylase activity could be recovered. Regarding the reutilization of the affinity adsorbents, α-amylase could be adsorbed and desorbed six times consecutively without a significant loss of α-amylase activity.     

Affinity binding of cells to cryogel adsorbents with immobilized specific ligands: effect of ligand coupling and matrix architecture

The capture of human acute myeloid leukemia KG-1 cells expressing the CD34 surface antigen and the fractionation of human blood lymphocytes were evaluated on polyvinyl alcohol (PVA)-cryogel beads and dimethyl acrylamide (DMAAm) monolithic cryogel with immobilized protein A. The affinity ligand (protein A) was chemically coupled to the reactive PVA-cryogel beads and epoxy-derivatized monolithic cryogels through different immobilization techniques and the binding efficiency of the cell surface receptors specific antibody-labeled cells to the gels/beads was determined. The binding of cells to monolithic cryogel was higher (90-95%) compared with cryogel beads (76%). B-lymphocytes, which bound to the protein A-cryogel beads, were separated from T-lymphocytes with yields for the two cell types 74 and 85%, respectively. About 91% of the bound B-cells could be recovered without significantly impairing their viability. Our results show differences in the percentage of cell-binding to the immunosorbents caused by ligand density, flow shear forces and bond strength between the cells and the affinity surface once distinct chemical coupling of protein A, size of beads, sequence of antibody binding to protein A adsorbents, morphology and geometry of surface matrices were compared.     

Identification of a polymeric β-cyclodextrin-binding peptide from a phage-displayed peptide library

In this work, a phage-displayed peptide library was applied to identification of β-cyclodextrin (CD)-binding peptide tag, capable of being combined to target peptides or proteins in a homogeneous way by established methods such as peptide synthesis and the recombinant DNA technique. Four enriched sequences were obtained after five rounds of biopanning against polymeric β-CD beads. One of the sequences showed high binding affinity to β-CD beads with a dissociation constant of approximately 7 × 10^–6 M. The β-CD-binding sequence was used for immobilization of a hepatitis C virus (HCV) antigenic peptide on β-CD beads. The functionalized β-CD beads were successfully used for immunoassay of anti-HCV antibody with a detection limit of 1 ng. These results demonstrate that the identified peptide sequence has the potential of being used as an affinity tag to β-CD-containing surfaces.     

Evaluation of cellulose-based biospecific adsorbents as a stationary phase for lectin affinity chromatography

Macroporous cellulose Granocel was evaluated as a matrix for the immobilization of two lectins Concanavalin A (ConA) (108 kDa) and Wheat Germ Agglutinin (WGA) (36 kDa). Two different methods were employed for the immobilization of the lectins via their protein moieties by a Schiff's bases reaction. One of them results in covalent coupling of the lectin directly to the support and the other gives the attachment through a long spacer arm which benefits the immobilization of voluminous ConA molecules. The adsorbents were characterized by the glycoproteins sorption recording adsorption kinetic data and isotherms. The adsorbents demonstrated high affinity to glycoproteins with a sorption capacity in the column up to 7.4 mg/ml support and a high recovery (up to 93%). The adsorption isotherms of glucose oxidase (GOD) onto ConA adsorbents reveals an adsorption behavior with high and low affinity binding sites. The dissociation constant K(d) of the ligand-sorbate complex is approximately 1 x 10(-6) and 0.4 x 10(-5)M, respectively. It was supposed that the second step is related to the sorption of solvated GOD onto already adsorbed GOD forming sorbate dimers.     

Preparation and evaluation of Ricinus communis agglutinin affinity adsorbents using polymeric supports

A practicable and efficient procedure for preparation of Ricinus communis agglutinin (RCA) affinity adsorbents has been developed. For immobilization of RCA two different polymer-based supports, Toyopearl and TSKgel (TosoHaas), were used. RCA has been successfully immobilized onto these supports with amounts of coupled ligand between 15 and 23 mg/g dry support and corresponding coupling yields of 69-93% (w/w). The prepared affinity adsorbents were characterized concerning their binding capacity for the glycoprotein asialofetuin (ASF) and accessibility of the ligand binding sites. The high accessibility of 80% showed that steric hindrance was negligible at the present ligand density. RCA-Toyopearl was successfully applied in affinity chromatography of glycoproteins indicating its high specificity. A long-term stability test proved no change in capacity for a period of at least 12 months. High-performance affinity chromatography (HPLAC) was carried out using RCA-TSKgel. Experimental results showed that the prepared adsorbents are suitable for selective separation of glycoproteins and oligosaccharides and therefore can be used for investigations of adsorption characteristics of glycoconjugates and for laboratory-scale preparations.     

Evaluation of two different Concanavalin A affinity adsorbents for the adsorption of glucose oxidase

Concanavalin A (Con A) was selected as ligand and thus immobilized onto two different supports, namely the polymeric Toyopearl and the inorganic silica, with the protection of its binding sites provided during the coupling procedure. The prepared Con A affinity adsorbents were then employed to evaluate their adsorption behaviour for the enzyme glucose oxidase (GOD). The immobilization kinetics showed that the immobilization of Con A on silica supports was much faster than that on Toyopearl supports, which could highly reduce the possibility of the denaturation of Con A. The optimal adsorption conditions for binding of GOD onto the ligand were determined in terms of the pH value and the ionic strength of the adsorption medium. The adsorption isotherms for binding GOD onto two Con A affinity adsorbents fitted well with the Langmuir equation. The maximum adsorption capacity q(m) of Toyopearl Con A and silica Con A were 7.9 mg/ml and 4.9 mg/ml, with a dissociation constant K(d) of 4.8 x 10(-7)M and 2.6 x 10(-6)M, respectively. Due to the less diffusive resistance, silica Con A showed both higher adsorption and desorption rates for GOD when compared with Toyopearl Con A. The nonspecific adsorption of GOD was less than 8% for both end-capped Toyopearl and silica supports. The dynamic adsorption of GOD for five times repeated processes showed a high stability for both prepared adsorbents. All the results indicate a good suitability of both Con A adsorbents for affinity adsorption of GOD.     

Studies on endotoxin removal mechanism of adsorbents with amino acid ligands

In this paper, a series of adsorbents with different amino acid ligands for endotoxin removal were prepared and endotoxin adsorption capacities (EAC) in aqueous solution were studied using an affinity column. The results showed that the property and structure of amino acid ligands have great influence on EAC. As the increasing of isoelectric point and polarity of amino acids ligands, EACs of the adsorbents increased. In addition, computer simulation method was employed to a further investigation on the interaction between endotoxins and ligands. Based on the results, some adsorbents were applied to remove endotoxin from endotoxemia rabbit's serum. Similar adsorption results were observed and the removal efficiency of adsorbents with Arg, Ser ligands is up to 78%.     

Performance of hydrophobic chromatography in purification of alpha-amylase

Hydrophobic ligands were introduced onto agarose beads, and the adsorption capacity of the beads was measured. The adsorption capacity increased with increase in the carbon number of the ligand, ionic strength of the buffer solution, and temperature. Crude alpha-amylase was purified with these hydrophobic adsorbents and the breakthrough and elution curves were estimated based on the mass transfer theory. Under strongly hydrophobic conditions, impurities contained in crude feeds and the lack of uniformity of packing caused by aggregation of beads affected adsorption and elution behaviors.     

Superparamagnetic adsorbents for high-gradient magnetic fishing of lectins out of legume extracts

This work presents the development, testing, and application in high-gradient magnetic fishing of superparamagnetic supports for adsorption of lectins. Various approaches were examined to produce affinity, mixed mode, and hydrophobic charge induction type adsorbents. In clean monocomponent systems affinity supports created by direct attachment of glucose or maltose to amine-terminated iron oxide particles could bind concanavalin A at levels of up to approximately 280 mg g(-1) support with high affinity ( approximately 1 microM dissociation constants). However, the best performance was delivered by adsorbents featuring coupled tentacular dextran chains displaying a maximum binding capacity of 238 mg g(-1) and a dissociation constant of 0.13 microM. Adsorbents derivatized with mixed mode or hydrophobic charge induction ligands likewise demonstrated very high capacities for both concanavalin A and Lens culinaris agglutinin (> or = 250 mg g(-1)) with dissociation constants in the micromolar range, though neither of these systems showed any selectivity for lectins in leguminous extracts. When the affinity supports were applied to carbohydrate containing legume extracts only the dextran-linked adsorbents supplied sufficient competition to dissolved sugars to selectively bind concanavalin A in an extract of jack beans. The dextran-linked supports were employed in a high-gradient magnetic fishing experiment, in which concanavalin A was purified to near homogeneity from a crude, unclarified extract of jack beans.     

The aqueous solubility and thermal stability of α-lipoic acid are enhanced by cyclodextrin

We investigated the effects of various cyclodextrins (CDs) on the aqueous solubility and thermal stability of α-lipoic acid, a compound with low water solubility. α-CD, β-CD, and γ-CD had little effect on the aqueous solubility of α-lipoic acid. In contrast, 6-O-α-maltosyl-CDs increased it in a concentration-dependent manner, 6-O-α-maltosyl-β-CD enhancing solubility the most. The thermal stability of α-lipoic acid in the solid state was improved by the addition of G2-β-CD(?), a commercial product of 6-O-α-maltosyl-β-CD. The thermal stability of α-lipoic acid was also improved by the addition of β-CD. Analysis by differential scanning calorimetry showed that G2-β-CD(?), a mixture of maltosyl-β-CDs, and β-CD efficiently formed complexes with α-lipoic acid. These results suggest that the sizes and shapes of these β-CD compounds are compatible with complexation with α-lipoic acid. Moreover, both the formation of an aqueous complex with G2-β-CD(?) and an insoluble complex with β-CD increased the thermal stability of α-lipoic acid.     

Intermolecular transglycosylating reaction of cyclodextrin glucanotransferase immobilized on capillary membrane

The intermolecular transglycosylating reaction of cyclodextrin glucanotransferase ([EC 2.4.1.19]; CGTase) immobilized on a capillary membrane was investigated using low molecular weight substrates such as cyclodextrin (CD), maltooligosaccharide (MOS), and a CD-MOS mixture. The immobilized CGTase catalyzed the conversion reaction of α-CD to β-CD and MOS or β-CD to α-CD and MOS within a short residence time. The conversion ratio increased as the amount of immobilized CGTase increased. The addition of glucose, maltose, and sucrose as acceptors in the substrate solution containing CD resulted in the acceleration of CD degradation compared with only CD substrate. Furthermore, the MOS substrate (degree of polymerization =2–6) was disproportionated with a conversion ratio exceeding 70% by the immobilized CGTase. These data demonstrate that immobilized CGTase can catalyze intermolecular transglycosylation between low molecular substrates in a few minutes by regulating the amount of immobilized enzyme and the residence time. This might contribute to our comprehension of CGTase-immobilized bioreactors for CD production as well as to the development of new glycosides through its excellent transglycosylation ability.     

Formation of Inclusion Complexes of Cycldextrin with Ethanol under Anhydrous Conditions

Complex formation of poorly water soluble organic compounds with cyclodextrin (CD) is quite difficult in an aqueous cyclodextrin system. Formation of the inclusion complex of d-limonene, phenyl ethanol, acetophenone, or menthol was investigated in a slurry form of α-, β-, or γ-CD in organic solvents or alcohol under anhydrous conditions. Ethanol and methanol were found to be good solvents for this method. The use of ethanol as the solvent was investigated in greater detail. There existed an optimal amount of ethanol for the maximum inclusion of d-limonene as the guest compound. However, an excess of ethanol inhibited the inclusion. An adsorption model of alcohol on CD, analogous to the substrate inhibition model of enzyme kinetics, could correlate the inclusion ratio with the amount of alcohol added to CD.     

Integrated enzyme purification and immobilization processes with immobilized metal affinity adsorbents

Silica-based immobilized metal affinity chromatography adsorbents with various ligand densities were prepared for the purification and immobilization of poly(His)-tagged d-hydantoinase (DHTase). An adsorbent with a ligand density of 13.0 μmol Cu²⁺/g gel exhibiting the optimal selectivity and a capacity of 1.4 mg/g gel toward the poly(His)-tagged enzyme was identified. The adsorbent was used for the one-step purification of His-tagged enzymes from crude cell lysate with a purity above 90%. The silica-based affinity adsorbents are particularly well suited for industrial scale operations due to their robustness. A packed-bed bioreactor with the DHTase-loaded adsorbents was used for the continuous conversion of d,l-p-hydroxyphenylhydantoin (d,l-HPH) to N-carbamoyl-d-hydroxyphenylglycine, an intermediate for the production of d-hydroxylphenylglycine. Under optimal conditions, 60 °C and pH 8.0, a conversion of 60% was obtained at a residence time of 30 min. Upon extended operation, the catalytic activity of the biocatalysts declined significantly due to enzyme leakage and enzyme denaturation. The extent of enzyme leakage can be attenuated by crosslinking with glutaraldehyde. In this study, we successfully demonstrate that a packed-bed bioreactor containing silica-based IMAC adsorbents can be used for the direct purification and immobilization of poly(His)-tagged enzymes for biotransformation.     

乳腺生物反应器表达的重组人乳铁蛋白的分离纯化及生物活性鉴定

以国产高交联度的快流速琼脂糖为基质,合成了不同配基密度的SP(Sulfopropyl,磺酸基)离子交换介质,建立了乳腺生物反应器表达重组人乳铁蛋白(Recombinant Human Lactoferrin,rHLF)的纯化方法。以溶菌酶为模型蛋白考察了不同配基密度离子交换介质的静态和动态吸附行为,结果表明介质具有良好的吸附性能。不同配基密度离子交换介质均可纯化得到rHLF,其中,高配基密度(0.24mol/L)的离子交换介质每毫升可以处理50mL rHLF牛乳,rHLF收率为86.5%,纯度为98.5%。圆二色谱的测定结果表明纯化的rHLF二级结构与天然人乳铁蛋白一致。生物学功能实验结果表明,rHLF的铁结合与释放活性与天然人乳铁蛋白相似,浓度为5g/L的rHLF对大肠杆菌的生长具有明显的抑制作用。     

Mechanically enforced bond dissociation reports synergistic influence of Mn2+ and Mg2+ on the interaction between integrin α7β1 and invasin

Integrins require the divalent ions magnesium and manganese for ligand recognition. Here we mechanically enforced bond dissociation to explore the influence of these ions on the mechanical strength of the specific bond between α(7) β(1) integrin and its pathologically relevant ligand invasin. Upon addition of these cations to the measurement buffer, we observe a pronounced increase in the force necessary to separate integrin and invasin coated beads. Both ions were found to work synergistically. With free invasin in the measurement buffer we furthermore observe that competitive blocking of binding sites overrides the increase in binding strength of individual beads. We show that this is due to a very strong dependence of bond affinity on divalent ions. Our study illustrates the importance of divalent ions for the regulation of force transmission by integrin ligand bonds on the molecular level.     

A new metal chelate affinity adsorbent for cytochrome C

We have prepared a novel metal-chelate adsorbent utilizing N-methacryloyl-L-histidine methyl ester (MAH) as a metal-chelating ligand. MAH was synthesized by using methacryloyl chloride and l-histidine methyl ester dihydrochloride. Spherical beads with an average diameter of 75-125 microm were produced by suspension polymerization of 2-hydroxyethyl methacrylate (HEMA) and MAH carried out in an aqueous dispersion medium. Then, Cu(2+) ions were chelated directly on the chelating beads. Cu(2+)-chelated beads were used in the adsorption of cytochrome c (cyt c) from aqueous solutions. The maximum cyt c adsorption capacity of the Cu(2+)-chelated beads (658.2 micromol/g Cu(2+) loading) was found to be 31.7 mg/g at pH 10 in phosphate buffer. The nonspecific cyt c adsorption on the naked PHEMA beads was 0.2 mg/g. Cyt c adsorption increased with increasing Cu(2+) loading. Cyt c adsorption capacity was demonstrated for the buffer types with the effects in the order phosphate > HEPES > MOPS > MES > Tris-HCl. Cyt c molecules could be adsorbed and desorbed five times with these adsorbents without noticeable loss in their cyt c adsorption capacity.     

Immobilization of protein ligands on new formyl-spacer-carriers for the preparation of stable and high capacity affinity adsorbents

New procedures to immobilize high concentrations of protein ligands by reductive amination on two types of formyl-carriers (I & II) having different spacer lengths were investigated in order to prepare stable and high-capacity adsorbents essential for efficient affinity chromatography. Formyl-carrier (I) was prepared by reductive amination with glutaraldehyde of the amino-carrier obtained on amination of an epoxy-activated carrier. Formyl-carrier (II) was prepared by sodium metaperiodate (NaIO4) treatment of a glyceryl-carrier obtained on hydrolysis of an epoxy-activated carrier. Especially high concentrations of protein ligands were immobilized on formyl-Sepharose 4B (I) under very mild conditions (pH 7.0, 4 degrees C). A series of lectins, one of the most useful classes of group-specific ligands, was successfully immobilized by the procedures. Concanavalin A-Sepharose 4B (I) thus obtained exhibited an adsorption capacity five times greater than that of concanavalin A-Sepharose 4B made by Pharmacia Fine Chemicals, and could be repeatedly used over twenty times without a significant reduction in its adsorption capacity.     

Liquid chromatographic studies with immobilized neuronal nicotinic acetylcholine receptor stationary phases: effects of receptor subtypes,pH and ionic strength on drug–receptor interactions

Nicotinic acetylcholine receptor (nAChR) α3-subunits, β4-subunits, α3/β4-subunit combination and α4/β2-subunit combination were immobilized on chromatographic stationary phases and the binding affinities of the different nAChR subtypes were chromatographically evaluated. The observed relative binding affinities of epibatidine were α4/β2>α3/β4 and epibatidine did not bind at α3-subunits and β4-subunits. No significant difference in binding affinities was observed on the α4/β2 nAChRs immobilized in immobilized artificial membrane (IAM) particles and those sterically immobilized on Superdex 200 beads. The effects of mobile phase pH and ionic strength on the binding affinities of the α3/β4 nAChRs support were also investigated. The results are consistent with the proposed ligand–nAChR binding model in which a cationic center exists at the binding site.