A general approach for antibody purification utilizing [Protein A-catechol:Fe3+] macro-complexes

A general platform for antibody purification utilizing free nonimmobilized Protein A modified with the strong metal chelator catechol (ProA-CAT) and Fe3+ ions is presented. The mechanism of purification requires formation and precipitation of macro-complexes composed of [ProA-CAT:IgG:Fe3+]. Target IgGs are eluted directly from the precipitates (i.e. pellets) at pH 3 in high yields (71-80%) and high purity (>95%), without dissociating the [ProA-CAT:Fe3+] insoluble macro-complex.     

Terminal alpha-linked galactose rather than N-acetyl lactosamine is ligand for bovine heart galectin-1 in N-linked oligosaccharides of glycoproteins

Preference for the beta-anomer of galactose attributed to the bovine heart 14 kDa galectin-1 (BHL-14) was re-examined using natural glycoproteins and artificially glycosylated proteins as ligands. Endogenous glycoproteins co-purified with BHL-14 during its affinity chromatographic isolation contained oligosaccharides bearing terminal alpha-linked galactose (TAG) moieties and were superior even to laminin as ligands for homogeneous BHL-14 obtained by high pressure liquid chromatography. Artificially glycosylated proteins prepared by covalent attachment of melibiose to proteins and containing TAG moieties were ligands for BHL-14, unlike their lactose counterparts which contained beta-linked galactose. Enzymatic removal of TAG moieties from the following glycoproteins abolished their recognition by BHL-14: (i) endogenous glycoproteins co-purified with BHL-14; (ii) mouse laminin; and (iii) bovine heart glycoproteins recognized by peanut agglutinin. Modification of TAG in laminin using galactose oxidase also rendered the glycoprotein inert towards BHL-14. Desialylation of human IgG, bovine thyroglobulin or laminin failed to increase the affinity of BHL-14 for these glycoproteins. Since removal of TAG or of sialic acid moiety exposed LacNAc (Gal beta1-->4 GlcNAc) in these glycoproteins, these results indicated that TAG, rather than LacNAc, is a ligand for BHL-14 on N-linked oligosaccharide chains of glycoproteins. Ready recognition of human IgA and jacalin-binding human plasma glycoproteins and non-recognition of human IgG suggested that T antigen (Galbeta1-->3 GalNAc) may also be ligand for galectin-1.     

Derivatized nanoparticle coated capillaries for purification and micro-extraction of proteins and peptides

Various methods are used to enrich or purify a protein of interest from other proteins and components in a crude cell lysate or other sample. One of the most powerful methods is affinity purification, also called affinity chromatography, whereby the proteins of interest are purified by virtue of their specific binding properties to an immobilized ligand. Affinity purification is becoming more widely used for exploring post-translation modifications and protein-protein interactions, especially with a view toward developing new general tag systems and strategies of chemical derivatization on peptides for affinity selection. Our work was aimed to immobilize proteins or ligands for affinity purification of antibodies, fusion-tagged proteins and other proteins and peptides. Selected proteins or peptides are efficiently extracted and enriched using chemically derivatized walls of a fused silica capillary column. In this paper, we present an open tubular capillary, where the inner wall of a fused silica capillary was derivatized by covalent binding of modified polystyrene latex particles. The capillaries were derivatized with iminodiacetic acid and loaded with Fe3+ or Ni2+ for the purification and enrichment of phosphopeptides or His-tagged proteins, respectively. The latex coated capillaries have been successfully applied to enrich phosphopeptides from beta-casein tryptic digest and ovalbumin tryptic digest at a micro volume scale with recoveries ranging from 92 to 95%. The capillaries have been eluted under conditions compatible with MALDI-MS without any prior desalting step. In another approach, concanavalin A (Con A) or Protein G were immobilized on the epoxy modified latex on the inner wall of the fused silica capillary for the purification of glycoproteins and immunoglobulin, respectively. The design of the capillary and the protocols used for purification permits the direct detection of eluted proteins and peptides with gel electrophoresis or with mass spectrometry. The elution volumes are passed as discrete segments of few microliters over the inner surface of the open-tube capillary, achieving enrichment factors of more than 20-fold from starting samples.     

Exploring variation in binding of Protein A and Protein G to immunoglobulin type G by isothermal titration calorimetry

Bacterial Protein A (PrtA) and Protein G (PrtG) are widely used for affinity purification of antibodies. An understanding of how PrtA and PrtG bind to different isotypes of immunoglobulin type G (IgG) and to their corresponding Fc fragments is essential for the development of PrtA and PrtG mimetic ligands and for the establishment of generic processes for the purification of various antibodies. In this paper, the interactions between the two IgG-binding proteins and IgG of two different subclasses, IgG1 and IgG4, as well as their analogous Fc fragments have been studied by isothermal titration calorimetry. The results indicate that both protein ligands bind IgG and Fc fragments strongly with Ka values in the range of 10(7) -10(8) M(-1) and for both ligands, the interaction with both IgG isotypes is enthalpically driven though entropically unfavorable. Moreover, variation in the standard entropic and standard enthalpic contribution to binding between the two isotypes as well as between IgG and Fc fragment implies that the specific interaction with PrtA varies according to IgG isotype. In contrast to PrtA, PrtG bound to F(ab')(2) fragment with a Ka value of 5.1 × 10(5) M(-1) ; thus underscoring the usefulness of PrtA as a preferred ligand for generic antibody purification processes.     

Purification of animal immunoglobulin G (IgG) using peptoid affinity ligands

The availability of highly pure animal antibodies is critical in the production of diagnostic tools and biosensors. The peptoid PL16, previously isolated from an ensemble of peptoid variants of the IgG-binding peptide HWRGWV, was utilized in this work as affinity ligand on WorkBeads resin for the purification of immunoglobulin G (IgG) from a variety of mammalian sources and chicken immunoglobulin Y (IgY). The chromatographic protocol initially optimized for murine serum and ascites was subsequently employed for processing rabbit, goat and sheep, donkey, llama, and chicken sera. The PL16-WorkBeads resin proved able to recover all antibody targets with values of yield between 50 and 90%, and purity consistently above 90%. Notably, PL16 not only binds a broader spectrum of animal immunoglobulins than the reference ligands Protein A and G, but it also binds equally well with all their subclasses. Unlike the protein ligands, in fact, PL16 afforded excellent values of yield and purity of mammalian polyclonal IgG, namely murine (47 and 94%), rabbit (66.5 and 91.7%), caprine IgG (63 and 91–95%), donkey, and llama (93 and 97%), as well as chicken IgY (42 and 92%). Of notice, it is also the ability of PL16 to target monomeric IgG without binding aggregated IgG; when challenged with a mixture of monomeric and aggregated murine IgG, PL16 eluted <3% of fed aggregates, against 11–13% eluted by Protein A and G. Collectively, these results prove the potential of the proposed peptoid ligand for large-scale purification of animal immunoglobulins.     

Concanavalin A-Sepharose Affinity Chromatography of Axon Plasma Membrane Proteins. Heterogeneity of Cholinergic Binding Sites

Abstract: Lysolecithin-solubilized proteins from axon plasma membranes of lobster walking leg nerve bundles were chromatographed on concanavalin A (Con A)-sepharose. Bound glycoproteins were eluted with α-methyl-D- mannoside. Near quantitative recovery of total protein was observed, 20–30% of the total protein being eluted in the Con A-binding glycoprotein fraction. A 5-fold enrichment of acetylcholinesterase (AChE) activity was achieved, demonstrating the glycoprotein nature of the axonal enzyme. The chromatographed fractions were characterized for binding of [³H]quinuclidinyl benzilate (QNB), [³nicotine (Nic), and [1251]α-bung arotoxin (BgTx) in an attempt to distinguish possible "muscarinic" and "nicotinic" binding sites in axonal membranes. All of the high-affinity "muscarinic" [³H]QNB binding activity appeared in the non-Con A-binding protein fractions, while binding of the two "nicotinic" ligands, [³Nic and ¹²⁵I-BgTx, was found in both the glycoprotein and non-Con A-binding protein fractions. BgTx interaction with the Con A-binding glycoproteins could be blocked with dtubocurarine, but BgTx binding in the non-Con A-binding proteins was not inhibited by curare. The significance of multiple cholinergic binding sites in axonal membranes is discussed. These data suggest a closer similarity between the cholinergic ligand binding proteins of peripheral nerve membrane and ganglia than between the axonal cholinergic binding sites and the ACh receptor of the neuromuscular junction.     

Nigral 6-hydroxydopamine lesions equally decrease mu and delta opiate binding to striatal patches: further evidence for a conformationally malleable type 1 opiate receptor

We have investigated the effect of nigral 6-hydroxydopamine (6-OHDA) lesions on binding of the mu receptor ligand dihydromorphine (DHM) and the delta receptor ligand [D-Ala2, D-Leu5]-enkephalin (DADLE) to sections of rat striatum under conditions which yield mu-like and delta-like ligand selectivities at discrete receptor patches (Type 1 receptor). 3H-DHM binding was decreased 43% while 3H-DADLE was decreased 22%. However, when the contribution of diffuse binding (Type 2) which is not affected by 6-OHDA is subtracted from the patch, the decrease is approximately 49% for both ligands. These data support the hypothesis that the Type 1 receptor of striatal patches is a conformationally malleable receptor entity which can exist in states having high affinities for various classes of opiate ligands.     

Novel sulfamethazine ligand used for one-step purification of immunoglobulin G from human plasma

To replace conventional affinity ligand like protein A or protein G, a pseudobioaffinity ligand seems to be an alternative for the purification of immunoglobulin G (IgG). In this study, sulfamethazine (SMZ) was chosen as novel affinity ligand for investigating its affinity to human IgG. Monodisperse, non-porous, cross-linked poly (glycidyl methacrylate) (PGMA) beads were employed as the support for high-performance affinity chromatography. SMZ was immobilized on PGMA beads using bisoxirane (ethanediol diglycigyl ether) as spacer. The resultant affinity media presented minimal non-specific interaction with other proteins. Results of high-performance frontal analysis indicated that the media showed specific affinity to human IgG with a dissociation constant on the order of 10(-6) M. The SMZ affinity column proved useful for a very convenient one-step purification of IgG from human plasma. Antibody purity after a one-step purification was higher than 90%, as determined by densitometric scanning of sodium dodecyl sulfate-polyacrylamide gel electrophoresis of purified fraction under reducing condition. The results obtained indicate that SMZ is a valuable affinity ligand for purification of human IgG.     

π-Bonded alkene and arene complexes of silver(I): an electrospray mass spectrometric study

Electrospray mass spectra have been observed for a number of alkene and arene complexes of Ag(I) formed by the interaction of AgNO₃ and the organometallic ligand in water/methanol solution. The ES mass spectra show that almost all the alkene and arene ligands in stoichiometric excess form labile 1:2 cationic complexes with Ag(I) which are easily decomposed by collisional activation to the 1:1 species. However, with a deficiency of organic ligand polymeric species are observed. The cation [Ag(cod)₂]⁺ (cod=1,5-cyclooctadiene) was reacted with a variety of other potential ligands, such as PPh₃, AsPh₃, PhSCH₂SPh etc. In most cases, mixed complexes [Ag(cod)(ligand)]⁺ were observed, and excess ligand usually produced [Ag(ligand)₂]⁺.     

Carbamoylphosphate serves as the source of CN(-), but not of the intrinsic CO in the active site of the regulatory [NiFe]-hydrogenase from Ralstonia eutropha

Within the catalytic centre of [NiFe]-hydrogenases one carbonyl and two cyanide ligands are covalently attached to the iron. To identify the metabolic origins of these ligands, the regulatory [NiFe] hydrogenase in conjunction with the indigenous Hyp maturation proteins of Ralstonia eutropha H16 were heterologously overproduced in E. coli grown in the presence of L-[ureido-(13)C] citrulline and NaH(13)CO(3). Infrared spectroscopy of purified hydrogenase provided direct evidence that only the cyanide ligands, but not the CO ligand, originate from CO(2) and carbamoylphosphate. Incorporation of label from (13)CO exclusively into the carbonyl ligand indicates that free CO is a possible precursor in carbonyl ligand biosynthesis.     

An artificial receptor for glycoproteins

We describe the rational design, synthesis and development of a sterilizable biomimetic ligand for the affinity purification of glycoproteins. Based on mimicking the principles of natural carbohydrate recognition, a putative library of 196 glycoprotein-binding synthetic ligands was designed and synthesized on a polymeric support. Ligand 11/11, based on a triazine scaffold and immobilized on a hydrophilic support, was identified as the "lead" ligand. The carbohydrate recognizing the potential of the "lead" ligand was revealed by reduced binding of a periodate oxidized model glycoprotein, and by "sharp" elution profiles achieved with borate buffer eluents. Specific elution and competitive binding experiments determined the monosaccharide specificity of 11/11 in the order mannoside > glucoside > galactoside. The diastereo-selective performance of ligand 11/11 was quantified and reaffirmed by analytical affinity chromatography and (1)H-NMR, in the order galactoside < glucoside < mannoside with binding affinities (K(a), M(-1)) in the 63-214 and 20-83 M(-1) range, respectively. Partition coefficient analysis revealed binding constants towards glycoproteins in the 10(4) M(-1) range, that compared favourably with the affinities of carbohydrate binding lectins for glycoproteins, such as concanavalin A. Molecular modelling studies of ligand 11/11 revealed the formation of a pre-organized apolar "tweezer-like" cavity, containing complementary nitrogenous hydrogen bond donor and acceptor groups that formed selective interactions with the equatorial 3- and 4-hydroxyl groups of saccharides.     

A novel application of thermo-responsive polymer to affinity precipitation of polysaccharide

The lectin receptors concanavalin A (ConA) and wheat germ lectin (WGL) were successfully conjugated to a thermo-responsive hydrogel polymer (poly-N-isopropylacrylamide) (PNIPAAm) as an affinity ligand. The coupling efficiencies of the ConA and WGL to PNIPAAm were 5.1% and 44%, respectively. These lectin receptor-polymer conjugates were then tested for their efficiency in purification of various polysaccharides or polysaccharide-containing compounds such as beta-glucan. Results indicated that these conjugates separated various polysaccharides from a complex mixture. The use of a thermo-responsive polymer in low-temperature purification of potentially heat-labile glycoproteins is advantageous. Additionally, other affinity ligands could be coupled to the polymer for separation of the respective bioactive molecules.     

A novel approach for affinity-based screening of target specific ligands: application of photoreactive D-glyceraldehyde-3-phosphate dehydrogenase

A novel application of the photoaffinity technique has been developed for the efficient discovery of small ligand and macromolecule interaction. The approach, photoaffinity capture, uses a photoreactive protein together with immobilized ligand for the rapid screening of competitive inhibitors. The set of photoreactive glyceraldehyde-3-phosphate dehydrogenase (photo-GAPDH) and immobilized dye ligand was prepared and examined as a model system. The photo-GAPDH was shown to efficiently capture the immobilized ligand. When nonimmobilized competitive ligands were included in the system, the capture was prevented in accordance with the affinity of the ligands. The present approach would provide an efficient tool for affinity-based screening of ligand libraries.     

Filamentous bacteriophage display of a bifunctional protein A::scFv fusion

Filamentous bacteriophage display is a powerful and widely used technology for the selection of affinity ligands. However, the commonly used phagemid systems result in the production of a population of phage of which those displaying the ligand of interest represent only a small proportion. Through simple dilution and nonspecific binding effects, the presence of large numbers of ligand-free phage reduces the likelihood that weak binders will be successfully selected from a ligand library. To provide a means of avoiding such problems, we have introduced an affinity handle into the phage that permits the purification of ligand-displaying phage. The IgG binding domains ofStaphylococcus ciureus protein A (SpA) were fused to a ligand (single chain Fv[scFv]) which is displayed as a fusion with the phage surface protein ApIII. Phage-displaying SpA were separated by affinity chromatography using immobilized human IgG from non-displaying phage and the purified phage were shown to possess functional scFv. Comparisons of fusion proteins in which either the scFv or the affinity handle occupied the amino terminus of the fusion protein showed that, whereas SpA function was unaffected by position, scFv function was compromised when the scFv did not occupy the amino terminus.     

Affinity chromatographic purification of human immunoglobulin a from chinese hamster ovary cell culture supernatant

Hexamer peptide ligand HWRGWV, initially screened from a solid phase combinatorial peptide library for immunoglobulins G (IgG) purification, is shown to also have potential for immunoglobulin A (IgA) purification. The determined dissociation constants for hIgA on HWRGWV resins at three different peptide densities from 0.11 to 0.55 meq/g fall in the range of 10^?6–10^?7 M, which are somewhat lower than those for hIgG. Although relatively low dynamic binding capacity (DBC) in the range of 9.2–16.8 mg IgA/mL resin at linear flow rates from 173 to 35 cm/h were obtained for IgA compared to IgG, the DBC value of HWRGWV for IgA is much greater than current commercially available affinity ligands. Although relatively lower binding affinity to secretory IgA compared to monomeric IgA was observed, the peptide ligand resins exhibit great potential for large‐scale purification of both human IgA and secretory IgA. Recoveries of 96.0% and 94.3%, and purities of 90.3% and 91.7% were achieved for human IgA and secretory IgA purification, respectively, from spiked Chinese hamster ovary cell culture supernatants without an extra afterwash step. Over 95% in purities were achieved for IgA and secretory IgA with an extra afterwash step; however, the recoveries would decrease at least 15% and 40% for IgA and secretory IgA, respectively. © 2012 American Institute of Chemical Engineers Biotechnol. Prog., 2013     

Endocytosis via galactose receptors in vivo : Ligand size directs uptake by hepatocytes and/or liver macrophages

We followed the intrahepatic binding and uptake of variously sized ligands with terminal galactosyl residues in rat livers. The ligands were administered to prefixed livers in binding studies and in vivo and in situ (serum-free perfused livers) in uptake studies. Gold sols with different particle diameters were prepared: 5 nm (Au5), 17 nm (Au17), 50 nm (Au50) and coated with galactose exposing glycoproteins (asialofetuin (ASF) or lactosylated BSA (LacBSA)). Electron microscopy of mildly prefixed livers perfused with LacBSA-Au5 in serum-free medium showed ligand binding to liver macrophages, hepatocytes and endothelial cells. Ligands bound to prefixed cell surfaces reflect the initial distribution of receptor activity: pre-aggregated clusters of ligands are found on liver macrophages, single particles statistically distributed on hepatocytes and pre-aggregated clusters of particles restricted to coated pits on endothelial cells. Ligand binding is prevented in the presence of 80 mM N-acetylgalactosamine (GalNAc), while N-acetylglucosamine (GlcNAc) is without effect. Electron microscopy of livers after ligand injection into the tail vein shows that in vivo uptake of electron-dense galactose particles by liver cells is size-dependent. Using a LacBSA-Au preparation with heterogeneous particle diameter (2.2-11.7 nm) we found that hepatocytes take up only ligands up to the size of 7.8 nm, whereas particles of all sizes available in this experiment are found in liver macrophages and endothelial cells. ASF-Au17 and LacBSA-Au17 are endocytosed by liver macrophages and endothelial cells, but not by hepatocytes. ASF-Au50 is taken up by liver macrophages only. In vivo uptake by liver macrophages is mediated by galactose-specific recognition as shown by inhibition with GalNAc. Some 52-65% inhibition was measured in in vivo experiments and 78% inhibition in in situ experiments. GlNAc showed no inhibitory effect. Furthermore, we measured uptake of [125J]ASF and of [125J]ASF adsorbed to Au17 by the different cell populations of rat livers in vivo. While the bulk of the molecular ligand is found in the hepatocyte fraction, the particulate ligand is located in the sinusoidal fraction.     

Selective precipitation and purification of monovalent proteins using oligovalent ligands and ammonium sulfate

This paper describes a method for the selective precipitation and purification of a monovalent protein (carbonic anhydrase is used as a demonstration) from cellular lysate using ammonium sulfate and oligovalent ligands. The oligovalent ligands induce the formation of protein-ligand aggregates, and at an appropriate concentration of dissolved ammonium sulfate, these complexes precipitate. The purification involves three steps: (i) the removal of high-molecular-weight impurities through the addition of ammonium sulfate to the crude cell lysate; (ii) the introduction of an oligovalent ligand and the selective precipitation of the target protein-ligand aggregates from solution; and (iii) the removal of the oligovalent ligand from the precipitate by dialysis to release the target protein. The increase of mass and volume of the proteins upon aggregate formation reduces their solubility, and results in the selective precipitation of these aggregates. We recovered human carbonic anhydrase, from crude cellular lysate, in 82% yield and 95% purity with a trivalent benzene sulfonamide ligand. This method provides a chromatography-free strategy of purifying monovalent proteins--for which appropriate oligovalent ligands can be synthesized--and combines the selectivity of affinity-based purification with the convenience of salt-induced precipitation.     

Polyclonal Antibodies to the Glycine Receptor Antagonist Strychnine

Polyclonal antibodies have been raised in rabbits against the glycine receptor antagonist strychnine, coupled through a 2-amino substituent to the antigenic protein key-hole limpet haemocyanin. Strychnine binding of the predominantly immunoglobulin G (IgG) class of antibodies was measured by incubation with [3H]strychnine, followed by adsorption of IgG onto Staphylococcus aureus cells and filtration through glass-fibre filters under vacuum. Only strychnine and structurally related alkaloids or derivatives were able to inhibit [3H]strychnine binding to the IgG. A significant rank correlation was found between the potencies of these compounds to inhibit [3H]strychnine binding to the antibodies and to the glycine receptor in mouse spinal cord membranes. In contrast, preincubation of strychnine antibodies with a variety of ligands at other neurotransmitter, drug, or hormone receptors in the CNS (at 10(-4) M) failed to inhibit binding significantly. The failure of glycine to inhibit strychnine antibody binding is consistent with previous suggestions that the recognition sites for this amino acid on the CNS receptor may be conformationally distinct from those for the antagonist alkaloid. Strychnine antibodies may now help in the identification and purification of possible endogenous ligands at this alkaloid binding site in the CNS.     

Influence of type of linkage and spacer on the interaction of beta-galactoside-binding proteins with immobilized affinity ligands

Affinity chromatography provides a powerful tool for isolation of carbohydrate-binding proteins. However, the choice of the ligand and spacer has an important impact on effectiveness. The influence of several different ligands on qualitative and quantitative aspects of the purification of two beta-galactoside-specific lectins has been evaluated. Sepharose was modified by coupling four types of neoglycoproteins (galactosylated or lactosylated bovine serum albumin with increasing sugar content) and two naturally occurring asialoglycoproteins at similar densities. Carbohydrate ligands at essentially equal density were made accessible to the lectins by seven commonly used methods. The yield of mistletoe lectin was high when lactosylated neoglycoproteins were used for separation. For these resins the sugar incorporation exceeded 10 sugar groups per protein carrier molecule. The yield was similarly high with the asialoglycoproteins and with lactose; the sugar was coupled to the resin as a p-aminophenyl derivative or by means of divinyl sulfone activation. An epoxy group in linkages of galactose or lactose decreased the binding capacity. A quantitatively similar degree of protein yields was obtained for the beta-galactoside-binding protein of bovine heart, although different proteins were obtained when neoglycoproteins were used as ligand. The nature of the affinity ligand in lectin purification can increase the yield and may also influence the profile of the carbohydrate-binding proteins.     

Improved methods for prepurification and detection of staphylococcal enterotoxin B from cell-free culture filtrate

An improved ELISA method for the detection of Staphylococcal Enterotoxin B (SEB) in protein A preparations is presented. Fab fragments were obtained by digestion with papain of anti-SEB IgG bound to SEB immobilized on Sepharose 4B. Anti-SEB and peroxidase-labeled Fab fragments from secondary antibodies were successfully used in a modified ELISA of SEB in protein A preparations. SEB-Sepharose was used repeatedly for the production of anti-SEB Fab fragments by papain digestion without loss of affinity. In addition, for the purification of SEB from crude culture filtrates, an initial step utilizing a combined heat and pH treatment for the removal of significant amounts of contaminating proteins without losses of toxin activity is presented. This pretreatment step yielded positive effects in further downstream processing considering both shortened time and an increase in total recovery.