Influence of different spacer arms on Mimetic Ligand™ A2P and B14 membranes for human IgG purification

Microporous membranes are an attractive alternative to circumvent the typical drawbacks associated to bead-based chromatography. In particular, the present work intends to evaluate different affinity membranes for antibody capture, to be used as an alternative to Protein A resins. To this aim, two Mimetic Ligands? A2P and B14, were coupled onto different epoxide and azide group activated membrane supports using different spacer arms and immobilization chemistries. The spacer chemistries investigated were 1,2-diaminoethane (2LP), 3,6-dioxa-1,8-octanedithiol (DES) and [1,2,3] triazole (TRZ). These new mimetic membrane materials were investigated by static and by dynamic binding capacity studies, using pure polyclonal human immunoglobulin G (IgG) solutions as well as a real cell culture supernatant containing monoclonal IgG(1). The best results were obtained by combining the new B14 ligand with a TRZ-spacer and an improved Epoxy 2 membrane support material. The new B14-TRZ-Epoxy 2 membrane adsorbent provided binding capacities of approximately 3.1mg/mL, besides (i) a good selectivity towards IgG, (ii) high IgG recoveries of above 90%, (iii) a high Pluronic-F68 tolerance and (iv) no B14-ligand leakage under harsh cleaning-in-place conditions (0.6M sodium hydroxide). Furthermore, foreseeable improvements in binding capacity will promote the implementation of membrane adsorbers in antibody manufacturing.     

An ellipsometric study of the antigen-antibody interaction in the interphase solid/solution area]

Ellipsometric studies have proved that monoclonal immunoglobulin G(IgG) against gamma-interferon (gamma-INF) and immunoglobulin fraction (Ig-fraction) of rabbit blood serum against human serum albumin (HSA) are adsorbed according to the Langmuir model on the surfaces of mirror plates of covalently modified gamma-INF or HSA, respectively. The maximum surface concentrations (Tmax) and equilibrium adsorption constants (K) for IgG and Ig-fraction are equal to 2.57 pmol/cm2 and 2 x 10(7) M-1, 3.3 mg/m2 and 0.1 cm3/micrograms, respectively. The additional treatment of gamma-INF modified surfaces with Tween-20 leads to an increase of K IgG ut to 2.7 x 10(-7) M-1 while Tmax decreases up to 1.12 pmol/cm2 which is conditioned by the blocking of protein non-specific binding sites. The role of specific and non-specific interactions of IgG and Ig-fraction with covalently immobilized antigens was studied at antibody-antigen mixture adsorption. The necessity to apply this method to quantitative determination of gamma-IHF and HSA in solutions was proved.     

Activating piezoelectric crystal surface by silanization for microgravimetric immunobiosensor application

The development of a microgravimetric immunobiosensor using a piezoelectric quartz crystal as a detector requires a stable and reproducible immobilization method for ligand binding. The method of silanization using 3-aminopropyltriethoxysilane (APTES) has been widely used for activating the carrier surface. In the present study, APTES deposition on a piezoelectric crystal surface was studied under various solvent conditions. A fluorescence method, using fluorescence isothiocyanate as a dye, was demonstrated for the quantification of amino groups on the silanized piezoelectric crystal surface. The optimum binding conditions of APTES deposition on a piezoelectric crystal surface were incorporated for the covalent immobilization of protein on the crystal surface in developing a stable and sensitive microgravimetric immunobiosensor. Determination of immunoglobulin G (IgG) concentration was performed using APTES modified piezoelectric crystals coated with protein G. The resonant frequency shift, resulting from the formation of protein G-IgG complex on the crystal surface, correlated with the concentration of IgG in the range 10 ng/ml to 0·1 mg/ml. The APTES modified, protein G coated crystals were found to be quite stable and did not show a significant loss of sensitivity even after 12 weeks of storage at 4°C in a desiccator.     

Immobilization of immunoglobulins on silica surfaces. Stability.

The development of new immunosensors based on surface-concentration-measuring devices requires a stable and reproducible immobilization of antibodies on well-characterized solid surfaces. We here report on the immobilization of immunoglobulin G (IgG) on chemically modified silica surfaces. Such surfaces may be used in various surface-oriented analytical methods. Reactive groups were introduced to the silica surfaces by chemical-vapour deposition of silane. The surfaces were characterized by ellipsometry, contact-angle measurements and scanning electron microscopy. IgG covalently bound by the use of thiol-disulphide exchange reactions, thereby controlling the maximum number of covalent bonds to the surface, was compared with IgG adsorbed on various silica surfaces. This comparison showed that the covalently bound IgG has a superior stability when the pH was lowered or incubation with detergents, urea or ethylene glycol was carried out. The result was evaluated by ellipsometry, an optical technique that renders possible the quantification of amounts of immobilized IgG. The results outline the possibilities of obtaining a controlled covalent binding of biomolecules to solid surfaces with an optimal stability and biological activity of the immobilized molecules.     

Optimised affinity purification of polyclonal antibodies from hyper immunised ovine serum using a synthetic Protein A adsorbent, MAbsorbent A2P

This report describes the applicability of a synthetic chromatography adsorbent for large-scale purification of polyclonal immunoglobulin G from hyper immunised ovine serum. Under optimised conditions, MAbsorbent A2P was shown to bind approximately 27 mg mL(-1) of ovine immunoglobulin from undiluted serum, with eluted IgG purities of >95%, minor levels of albumin (approximately 1%) and undetectable levels of leached ligand in the purified preparations. The results presented here indicate that the optimised affinity capture of immunoglobulin from ovine serum using MAbsorbent A2P is a feasible alternative to Protein A chromatography or sodium sulphate precipitation for the initial capture of antibodies from undiluted serum.     

Gum Arabic coated magnetic nanoparticles with affinity ligands specific for antibodies

A novel magnetic support based on gum Arabic (GA) coated iron oxide magnetic nanoparticles (MNP) has been endowed with affinity properties towards immunoglobulin G (IgG) molecules. The success of the in situ triazine ligand synthesis was confirmed by fluorescence assays. Two synthetic ligands previously developed for binding to IgG, named as ligand 22/8 (artificial Protein A) and ligand 8/7 (artificial Protein L) were immobilized on to MNPs coated with GA (MNP_GA). The dimension of the particles core was not affected by the surface functionalization with GA and triazine ligands. The hydrodynamic diameters of the magnetic supports indicate that the coupling of GA leads to the formation of larger agglomerates of particles with about 1 µm, but the introduction of the triazine ligands leads to a decrease on MNPs size. The non‐functionalized MNP_GA bound 28 mg IgG/g, two times less than bare MNP (60 mg IgG/g). MNP_GA modified with ligand 22/8 bound 133 mg IgG/g support, twice higher than the value obtained for ligand 8/7 magnetic adsorbents (65 mg/g). Supports modified with ligand 22/8 were selected to study the adsorption and the elution of IgG. The adsorption of human IgG on this support followed a Langmuir behavior with a Q_máx of 344 mg IgG/g support and K_a of 1.5 × 10⁵ M. The studies on different elution conditions indicated that although the 0.05 M citrate buffer (pH 3) presented good recovery yields (elution 64% of bound protein), there was occurrence of iron leaching at this acidic pH. Therefore, a potential alternative would be to elute bound protein with a 0.05 M glycine‐NaOH (pH 11) buffer. Copyright © 2010 John Wiley & Sons, Ltd.     

Purification of immunoglobulins G by protein A/G affinity membrane chromatography

An affinity membrane grafted with protein A/G or protein A was characterized for human and mouse immunoglobulins G purification. Breakthrough curves up to ligand saturation were measured and used to study the effects of flow velocities, feed solution concentrations and protein A/G versus protein A membranes. Increased flow-rate did not decrease the amount of IgG bound to the membranes. Increased feed solution concentration allowed more IgG to bind prior to breakthrough. Kinetic parameters for immunoglobulins G sorption to immobilized protein A were measured in batch experiments. The static binding capacity was determined to be 6.6 mg ml⁻¹ membrane volume. Finally, this affinity membrane was used to purify IgG from cell culture supernatant. The electrophoresis of the purified IgG fractions did not show any contaminant.     

Fabrication and characterization of 3D hydrogel microarrays to measure antigenicity and antibody functionality for biosensor applications

We report the fabrication, characterization and evaluation of three-dimensional (3D) hydrogel thin films used to measure protein binding (antigenicity) and antibody functionality in a microarray format. Protein antigenicity was evaluated using the protein toxin, staphylococcal enterotoxin B (SEB), as a model on highly crosslinked hydrogel thin films of polyacrylamide and on two-dimensional (2D) glass surfaces. Covalent crosslinking conditions were optimized and quantified. Interrogation of the modified 3D hydrogel was measured both by direct coupling of a Cy5-labeled SEB molecule and Cy5-anti-SEB antibody binding to immobilized unlabeled SEB. Antibody functionality experiments were conducted using three chemically modified surfaces (highly crosslinked polyacrylamide hydrogels, commercially available hydrogels and 2D glass surfaces). Cy3-labeled anti-mouse IgG (capture antibody) was microarrayed onto the hydrogel surfaces and interrogated with the corresponding Cy5-labeled mouse IgG (antigen). Five different concentrations of Cy5-labeled mouse IgG were applied to each microarrayed surface and the fluorescence quantified by scanning laser confocal microscopy. Experimental results showed fluorescence intensities 3-10-fold higher for the 3D films compared to analogous 2D surfaces with attomole level sensitivity measured in direct capture immunoassays. However, 2D surfaces reported equal or greater sensitivity on a per-molecule basis. Reported also are the immobilization efficiencies, inter-and intra-slide variability and detection limits.     

Cross-linking of the high affinity Fc receptor for human immunoglobulin G1 triggers transient activation of NADPH oxidase activity. Continuous oxidase activation requires continuous de novo receptor cross-linking

Cross-linking of the high affinity Fc receptor for human immunoglobulin G1 (Fc gamma RI) on U937 cells triggered superoxide anion (O-2) release. This was accomplished by the binding of an Fc gamma RI-specific monoclonal antibody, mAb 32, followed by cross-linking of the mAb on the cell with anti-mouse IgG F(ab')2 by Fc gamma RI-specific mAbs 32 and 22 used as an equimolar mixture or by Fc gamma RI-specific mAb 197 (a murine IgG2a and thus a multivalent ligand for Fc gamma RI) alone. At subsaturating concentrations of the Fc gamma RI-cross-linking ligands, O2- generation was continuous over relatively long intervals. However, saturating concentrations triggered an often substantial but always transient O2- burst. This transient burst of oxidase activity ceased with maximal ligand accumulation on the cell. Cells in which oxidase activity had ceased could be restimulated using phorbol 12-myristate 13-acetate or aggregated human IgG1, indicating that cessation of O2- generation was not due to a generalized exhaustion or inhibition of the NADPH oxidase pathway. Cells incubated in subsaturating concentrations of cross-linking antibodies continued to release O2- until binding of the ligand ceased. In addition, the rates of O2- production and ligand accumulation were the same. Thus, continuous O2- production appeared to be dependent upon continuous de novo formation of cross-linked and activated Fc gamma RI. Furthermore, the mol of O2- released in response to Fc gamma RI cross-linking by the multivalent ligand mAb 197 were directly proportional to the mol of mAb bound over a range of saturating and subsaturating concentrations. This evidence suggests a quantal relationship between each Fc gamma RI activated (cross-linked) and the resultant oxidase activity and supports a "rate" model for the activation of this response. Thus, each Fc gamma RI entering the pool of activated receptors probably makes a unitary contribution to the signal. An additional finding showed that cross-linked Fc gamma RI became associated with the cell cytoskeleton and that this association was also transient. Dissociation of Fc gamma RI from its cytoskeletal attachment occurred well after cessation of O2- production.     

Application of dye-ligands affinity adsorbent in capturing of rabbit immunoglobulin G

The applicability of dye-ligands attached to an expanded bed chromatography quartz base matrix (Streamline™) for the affinity bioseparation of rabbit immunoglobulin G (IgG) was investigated. Reactive Green 5 (RG-5) immobilized onto adsorbent was selected for capturing of rabbit-IgG due to its higher binding capacity compared to other dye-ligands possessing similar ligand density. Adsorption parameters such as pH, temperature, ionic strength and initial rabbit-IgG concentration were optimized for the adsorption of rabbit-IgG on the RG-5-immobilized adsorbent. The highest rabbit-IgG adsorption was recorded in pH 7.0, while the maximum binding capacity for BSA was achieved at pH 4.0. The adsorption of rabbit-IgG on RG-5-immobilized adsorbent was declined as the increase of ionic strength. There is no significant influence of temperature against adsorption efficiency of RG-5-immobilized adsorbent for rabbit-IgG. The adsorption phenomenon of rabbit-IgG on RG-5-immobilized adsorbent appeared to follow the Langmuir–Freundlich adsorption isotherm model. The theoretically maximum binding capacity (q_m) of RG-5-immobilized adsorbent estimated from this isotherm was 49.3 mg ml⁻¹, which is very close to that obtained experimentally (49.0 mg ml⁻¹). About 50% of bound BSA on RG-5-immobilized adsorbent in binary adsorption system was removed with washing buffer containing 1 M NaCl.     

Infrared evidence of azide binding to iron, copper, and non-metal sites in heart cytochrome c oxidase.

Interactions of azide ion with bovine heart cytochrome c oxidase (CcO) at five redox levels (IV) to (0), obtained by zero to four electron reduction of fully oxidized enzyme CcO(IV), were monitored by infrared and visible/Soret spectra. Partially reduced CcO gave three azide asymmetric stretch band at 2040, 2016, and 2004 cm-1 for CcO(III)N3 and two at 2040 and 2016 cm-1 for CcO(II)N3 and CcO(I)N3. Resting CcO(IV) reacts with N3- to give one band at 2041 cm-1 assigned to CuB2+N3 and another at 2051 cm-1 to N3- that is associated with protein but is not bound to a metal ion. At high azide concentrations the weak association of many azide molecules with non-metal protein sites was observed at all redox levels. These findings provide direct evidence for 1) N3- binding to CuB as well as Fea3 in partially reduced enzyme, but no binding to Fea3 in fully oxidized enzyme and no binding to either metal in fully reduced enzyme; 2) a long range effect of the oxidation state of Fea or CuA on ligand binding at heme a3, but not at CuB; and 3) an insensitivity of either Fea3 or CuB ligand site to changes in ligand or oxidation state at the other site. The observed independence of the Fea3 and CuB sites provides further support for Fea3(3)+ OOH, rather than Fea3(3)+ OOCuB2+, as an intermediate in the reduction of O2 to water by the oxidase.     

Independent effects of IgG and complement upon human polymorphonuclear leukocyte function.

Particle ingestion by polymorphonuclear leukocytes (PMN) is promoted by cell surface recognition and binding of fragments of the third component of complement (C3) and Fc regions of certain immunoglobulin (IgG) molecules. In order to determine the influence of these specific ligandsurface membrane interactions upon other PMN functions, we have employed nonphagocytosable particles (serum-treated Sepharose beads) coated with fragments of C3 and/or IgG, and have investigated whether these provide a sufficient stimulus for the metabolic changes and degranulation that ordinarly accompany phagocytosis by PMN. Sepharose 4B activates complement in fresh normal serum and consequently is coated with fragments of C3 (confirmed by immunoelectrophoretic evidence of factor B and C3 conversion and by immunofluorescence). Adsorbed IgG could be removed from serum-treated Sepharose by boiling in 2 M NaCl without significantly influencing bound complement. We have found that normal human PMN recognize and adhere to Sepharose beads coated with fragments of C3 and consequently are stimulated to increase their oxidative metabolism (measured as superoxide anion generation). This PMN response occurred in the absence of IgG but could be amplified if this immunoglobulin was also present on the bead surfaces.Both adherence and metabolic stimulation could be blocked by treatment of the beads with F(ab)2 anti-C3. In contrast to metabolic stimulation, degranulation (selective extracellular release of lysosomal constituents) was observed only when PMN encountered both C3 fragments and IgG on the beads. This response could be blocked by treating beads with either F(ab)2 anti-C3 or F(ab)2 anti-IgG. These results indicate that cell surface stimulation of PMN is not an "all or none" phenomenon and that certain vital functions of these cells may be mediated or modulated independently by immunoglobulins and complement.     

Two sites of interaction of anions with cytochrome a in oxidized bovine cytochrome c oxidase

An interaction between cytochrome a in oxidized cytochrome c oxidase (CcO) and anions has been characterized by EPR spectroscopy. Those anions that affect the EPR g = 3 signal of cytochrome a can be divided into two groups. One group consists of halides (Cl-, Br-, and I-) and induces an upfield shift of the g = 3 signal. Nitrogen-containing anions (CN-, NO2-, N3-, NO3-) are in the second group and shift the g = 3 signal downfield. The shifts in the EPR spectrum of CcO are unrelated to ligand binding to the binuclear center. The binding properties of one representative from each group, azide and chloride, were characterized in detail. The dependence of the shift on chloride concentration is consistent with a single binding site in the isolated oxidized enzyme with a Kd of approximately 3 mm. In mitochondria, the apparent Kd was found to be about four times larger than that of the isolated enzyme. The data indicate it is the chloride anion that is bound to CcO, and there is a hydrophilic size-selective access channel to this site from the cytosolic side of the mitochondrial membrane. An observed competition between azide and chloride is interpreted by azide binding to three sites: two that are apparent in the x-ray structure plus the chloride-binding site. It is suggested that either Mg2+ or Arg-438/Arg-439 is the chloride-binding site, and a mechanism for the ligand-induced shift of the g = 3 signal is proposed.     

Bovine Immunoglobulin/Protein Isolate Binds Pro-Inflammatory Bacterial Compounds and Prevents Immune Activation in an Intestinal Co-Culture Model

Intestinal barrier dysfunction is associated with chronic gastrointestinal tract inflammation and diseases such as IBD and IBS. Serum-derived bovine immunoglobulin/protein isolate (SBI) is a specially formulated protein preparation (>90%) for oral administration. The composition of SBI is greater than 60% immunoglobulin including contributions from IgG, IgA, and IgM. Immunoglobulin within the lumen of the gut has been recognized to have anti-inflammatory properties and is involved in maintaining gut homeostasis. The binding of common intestinal antigens (LPS and Lipid A) and the ligand Pam3CSK4, by IgG, IgA, and IgM in SBI was shown using a modified ELISA technique. Each of these antigens stimulated IL-8 and TNF-α cytokine production by THP-1 monocytes. Immune exclusion occurred as SBI (≤50 mg/mL) bound free antigen in a dose dependent manner that inhibited cytokine production by THP-1 monocytes in response to 10 ng/mL LPS or 200 ng/mL Lipid A. Conversely, Pam3CSK4 stimulation of THP-1 monocytes was unaffected by SBI/antigen binding. A co-culture model of the intestinal epithelium consisted of a C2BBe1 monolayer separating an apical compartment from a basal compartment containing THP-1 monocytes. The C2BBe1 monolayer was permeabilized with dimethyl palmitoyl ammonio propanesulfonate (PPS) to simulate a damaged epithelial barrier. Results indicate that Pam3CSK4 was able to translocate across the PPS-damaged C2BBe1 monolayer. However, binding of Pam3CSK4 by immunoglobulins in SBI prevented Pam3CSK4 translocation across the damaged C2BBe1 barrier. These results demonstrated steric exclusion of antigen by SBI which prevented apical to basal translocation of antigen due to changes in the physical properties of Pam3CSK4, most likely as a result of immunoglobulin binding. This study demonstrates that immunoglobulins in SBI can reduce antigen-associated inflammation through immune and steric exclusion mechanisms and furthers the mechanistic understanding of how SBI might improve immune status and reduce inflammation in various intestinal disease states.     

Internalization and degradation of macrophage Fc receptors bound to polyvalent immune complexes

We have studied the Fc receptor-mediated pinocytosis of immunoglobulin G (IgG)-containing immune complexes by mouse macrophages. IgG complexes were formed from affinity-purified rabbit dinitrophenyl IgG and dinitrophenyl modified BSA at molar ratios of 2.5-10:1. Both the specificity of binding and the fate of internalized receptors were analyzed using monoclonal and polyclonal anti-Fc receptor antibodies. Based on the susceptibility of surface-bound ligand to release by proteolysis, we have found that at 37 degrees C, 125I-labeled IgG complexes were rapidly internalized (t1/2 less than 2 min) and delivered to lysosomes; acid-soluble 125I was detectable in the growth medium within 5-10 min of uptake. However, kinetic evidence indicated that Fc receptors were not efficiently re-used for multiple rounds of ligand uptake. Instead, macrophages that were exposed continuously to saturating concentrations of IgG complexes exhibited a selective and largely irreversible removal of Fc receptors from the plasma membrane. This loss of surface receptors correlated with an increased rate of receptor turnover, determined by immune precipitation of Fc receptors from 125I-labeled macrophages. Thus, in contrast to the results obtained in the accompanying paper (I. Mellman, H. Plutner, and P. Ukkonen, 1984, J. Cell Biol. 98:1163-1169) using a monovalent ligand, these data indicate that the interaction of Fc receptors with polyvalent complexes leads to the degradation of both ligand and receptor following their delivery to lysosomes.     

Incorporation of membrane-anchored flagellin into influenza virus-like particles enhances the breadth of immune responses

We have designed a membrane-anchored form of the Toll-like receptor 5 ligand flagellin, the major proinflammatory determinant of enteropathogenic Salmonella, which was found to be glycosylated and expressed on cell surfaces. A chimeric influenza virus-like particle (cVLP) vaccine candidate containing A/PR8/34 (H(1)N(1)) hemagglutinin (HA), matrix protein (M1), and the modified flagellin as a molecular adjuvant was produced. The immunogenicity, including the serum antibody levels and cellular immune responses, and the protective efficacy against homologous and heterologous live virus challenge of the resulting VLPs were tested after intramuscular administration in a mouse model. The results demonstrated that flagellin-containing VLPs elicited higher specific immunoglobulin G (IgG) responses than standard HA and M1 VLPs, indicating the adjuvant effect of flagellin. Enhanced IgG2a and IgG2b but not IgG1 responses were observed with flagellin-containing VLPs, illuminating the activation of Th1 class immunity. The adjuvant effects of flagellin were also reflected by enhanced specific cellular responses revealed by the secretion of cytokines by freshly isolated splenocyte cultures when stimulated with pools of major histocompatibility complex class I or II peptides. When immunized mice were challenged with homologous live PR8 virus, complete protection was observed for both the standard and cVLP groups. However, when a heterosubtypic A/Philippines (H(3)N(2)) virus was used for challenge, all of the standard VLP group lost at least 25% of body weight, reaching the experimental endpoint. In contrast, for the cVLP group, 67% of mice survived the challenge infection. These results reveal that cVLPs designed by incorporating flagellin as a membrane-anchored adjuvant induce enhanced cross-protective heterosubtypic immune responses. They also indicate that such cVLP vaccines are a promising new approach for protection against pandemic influenza viruses.     

Beta-glycosylamidine as a ligand for affinity chromatography tailored to the glycon substrate specificity of beta-glycosidases

An affinity adsorbent for beta-glycosidases has been prepared by using beta-glycosylamidine as a ligand. beta-Glucosylamidine and beta-galactosylamidine, highly potent and selective inhibitors of beta-glucosidases and beta-galactosidases, respectively, were immobilized by a novel one-pot procedure involving the addition of a beta-glycosylamine and 2-iminothiolane.HCl simultaneously to a matrix modified with maleimido groups via an appropriate spacer to give an affinity adsorbent for beta-glucosidases and beta-galactosidases, respectively. This one-pot procedure enables various beta-glycosylamidine ligands to be formed and immobilized conveniently according to the glycon substrate specificities of the enzymes. A crude enzyme extract from tea leaves (Camellia sinensis) and a beta-galactosidase from Penicillium multicolor were chromatographed directly on each affinity adsorbent to give a beta-glucosidase and a beta-galactosidase to apparent homogeneity in one step by eluting the column with glucose or by a gradient NaCl elution, respectively. The beta-glucosidase and beta-galactosidase were inhibited competitively by a soluble form of the corresponding beta-glycosylamidine ligand with an inhibition constant (K(i)) of 2.1 and 0.80 microM, respectively. Neither enzyme was bound to the adsorbent with a mismatched ligand, indicating that the binding of the glycosidases was of specific nature that corresponds to the glycon substrate specificity of the enzymes. The ease of preparation and the selective nature of the affinity adsorbent should promise a large-scale preparation of the affinity adsorbent for the purification and removal of specific glycosidases according to their glycon substrate specificities.     

Lectin specificity and binding characteristics of human C-reactive protein.

C-reactive protein (CRP) is thought to play an important role in immunomodulation. The exact biologic function of this pentraxin protein is, however, still unclear. Here we report experiments designed to further characterize the binding properties of CRP. Using purified human CRP it could be shown that CRP immobilized onto polystyrene surfaces or onto latex beads binds distinct plasma glycoproteins including IgG, asialofetuin, asialo-beta 2-glycoprotein I and, likewise, synthetic glycoproteins as a lectin, exhibiting binding specificity for terminal galactosyl residues of the glycoprotein glycans. Binding of CRP to IgA, IgM, IgG, asialofetuin, asialo-beta 2-glycoprotein I and to synthetic glycoproteins requires immobilization onto surfaces of both CRP and the ligand. Fibronectin and fibrinogen are bound by surface-immobilized CRP also in soluble phase. Comparing various mono-, di-, and trisaccharides as competitive inhibitors of the lectin binding activity of CRP, only beta-D-Gal-(1-3)-D-GalNAc, beta-D-Gal-(1-4)-D-GalNAc, and beta-D-Gal-(1-4)-beta-D-Gal-(1-4)-D-GlcNAc had significant inhibitory power at a concentration of 8 mmol/liter. Binding activity of CRP was pH-dependent with an optimum at pH 5 to 6 and was reduced by 90% when pH was shifted from 6 to the physiologic pH value of 7.4. CRP exhibited lectin-like properties with binding specificity for galactosyl residues also when bound to K-562 erythroleukemia cells. It is therefore suggested that CRP immobilized onto surfaces exhibits lectin activity toward galactosyl groups preferentially in a mildly acidic environment as present at sites of inflammation.     

Crystal structure at 1.9 A resolution of the apoovotransferrin N-lobe bound by sulfate anions: implications for the domain opening and iron release mechanism

Several lines of functional evidence have shown that anion binding to a nonsynergistic site is a prerequisite for the anion-mediated iron release mechanism of transferrins. We report here structural evidence of the location of sulfate anion binding sites of the ovotransferrin N-lobe via the 1.90 A resolution apo crystal structure. The crystals were grown in an ammonium sulfate solution and belonged to space group P6(3)22 with the following unit cell dimensions: a = b = 125.17 A and c = 87.26 A. The structural determination was performed by isomorphous replacement, using Pt and Au derivatives. The structure refinement gave an R-factor of 0.187 in the resolution range of 7.0-1.90 A for the final model. From the electron density map, the existence of four bound SO(4)(2)(-) anions was detected. Three of them that exhibited reasonably low B-factors were all located in the opened interdomain cleft (sites 1-3). In site 1, the bound anion directly interacts with an Fe(3+)-coordinating ligand; SO(4) O1 and SO(4) O3 form hydrogen bonds with His250 NE2. Oxygen atom O3 of the same sulfate anion makes a hydrogen bond with Ser91 OG in a hinge strand. The sulfate anion in site 2 partially occupies the synergistic anion binding sites; SO(4) O2 and SO(4) O3 are hydrogen bonded to Arg121 NE and NH2, respectively, both of which are consensus anchor groups for CO(3)(2)(-) anion in holotransferrins. The former oxygen atom of SO(4)(2)(-) is also hydrogen bonded to Ser122 N, which forms a hydrogen bond with Fe(3+)-coordinating ligand Asp60 OD2 in holotransferrins. Some of the SO(4)(2)(-) oxygen atoms in sites 1 and 2 interact indirectly through H(2)O molecules with functionally important protein groups, such as the other Fe(3+)-coordinating ligands, Tyr92 OH and Tyr191 OH, and a dilysine trigger group, Lys209 NZ. In site 3, SO(4) O1 and SO(4) O4 form hydrogen bonds with Ser192 OG and Tyr191 N, respectively, and SO(4) O2 forms hydrogen bonds with Ser192 N and Ser192 OG. These structural data are consistent with the view that the anion bindings to the interdomain cleft, especially to sites 1 and 2, play crucial roles in the domain opening and synergistic carbonate anion release in the iron release mechanism of the ovotransferrin N-lobe.     

A biomimetic Protein G affinity adsorbent: an Ugi ligand for immunoglobulins and Fab fragments based on the third IgG‐binding domain of Protein G

This work reports the development of a synthetic affinity adsorbent for immunoglobulins based on the Fab‐binding domain of Streptococcal Protein G (SpG‐domain III). The ligand (A2C7I1) was synthesized by the four‐component Ugi reaction to generate a substituted peptoidal scaffold mimicking key amino acid residues of SpG. Computer‐aided analysis suggests a putative binding site on the C_H1 domain of the Fab molecule. In silico studies, supported by affinity chromatography in comparison with immobilized SpG, as well as analytical characterization by liquid chromatography/electrospray ionization–mass spectrometry and ¹H nuclear magnetic resonance of the ligand synthesized in solution, indicated the authenticity and suitability of the designed ligand for the purification of immunoglobulins. The immobilized ligand displayed an apparent static binding capacity of ~17 mg IgG ml^?1 and a dissociation constant of 5.34 × 10^?5 M. Preparative chromatography demonstrated the ability of the immobilized ligand to purify IgG and Fab fragments from crude mammalian and yeast cell cultures, under near physiological ionic strength and pH, to yield proteins of 99% and 93% purity, respectively. Copyright © 2013 John Wiley & Sons, Ltd.