Purification of wheat germ agglutinin by affinity chromatography

Wheat germ agglutinin was isolated in pure form and in high yield from an extract of wheat germ by affinity chromatography of 6-amino-1-hexyl-2-deoxy-β-d-glucopyranoside-Sepharose 4B. The purified agglutinin behaved as a single species electrophoretically and, as judged by its migration on sodium dodecyl sulfate-polyacrylamide gels, has an apparent molecular weight of 17,000. The amino acid composition of the isolated agglutinin was in good agreement with that previously reported.     

Purification of wheat germ agglutinin using affinity chromatography on chitin

Chitin, the naturally occurring polymer of N-acetyl-glucosamine, has been used as a ligand matrix for affinity chromatography of wheat germ agglutinin in a new purification method which is well suited for large scale preparations. The agglutinin obtained is homogeneous with respect to polypeptide chain molecular weight, has a blocked amino terminus and is free of proteolytic and β-N-acetyl-glucosaminidase activities.     

Purification of wheat germ agglutinin by affinity chromatography on a sepharose-bound N-acetylglucosamine derivative

Sepharose-2-acetamido-N-(?-aminocaproyl)-2-deoxy-β-D-glucopyranosylamine was prepared by a reaction of 2-acetamido-3,4,6-tri-0-acetyl-2-deoxy-β-D-glucopyranosylamine and N-(benzyloxycarboxyl)-?-aminocaproic acid, removal of the 0-acetyl and the benzyloxycarboxyl groups and coupling to Sepharose. The product was used for the purification of wheat germ agglutinin, by adsorption from a crude wheat germ extract and elution with 0.1M acetic acid. The purified agglutinin was homogeneous on SDS-polyacrylamide gel electrophoresis and had a specific hemagglutinating activity of 3000 u/mg when tested on trypsinized rabbit erythrocytes. It was rich in cysteine, cystine and glycine, and contained no sugar.     

A new affinity sorbent for the purification of lectins and its use in the purification of wheat germ agglutinin

A method is developed for obtaining gel from eggwhite and its application as a sorbent for purification of lectins. Eggwhite was treated by 1% glutaraldehyde at pH 5, for 5-6 hours at room temperature, then it was minced and washed by water. The residual aldehyde groups were blocked by glycine treatment. The sorbent obtained possessed high affinity for lectins specific to N-acetylglucosamine and complex oligosaccharides. The galactose- and mannose-specific lectins were adsorbed to a less extent. The purification of the wheat germ agglutinin using the eggwhite gel is described.     

Purification of pili and outer membrane vesicles of Neisseria gonorrhoeae by wheat germ agglutinin affinity chromatography

Previous studies indicated that OMVs of Neisseria gonorrhoeae reacted specifically with WGA. A technique was therefore developed for the separation of gonococcal pili and OMVs by WGA affinity chromatography. This method was more convenient and more effective than isopycnic centrifugation for obtaining OMVs free of pili. The antigens were further purified but though homogeneous as judged by in vitro analytical methods, they both elicited an antibody response in animals to minor impurities previously undetected.     

Protein composition of the atrial muscarinic acetylcholine receptor partially purified by wheat germ agglutinin affinity chromatography

The use of affinity chromatography on wheat germ agglutinin columns to partially purify detergent extracts of muscarinic acetylcholine receptor from porcine atria is described. The procedure results in a 20-fold purification of the protein. The partially purified protein binds [3H]L-QNB (the L isomer of quinuclidinyl benzilate) with an observed association rate constant 2- to 3-fold lower than that found for the detergent extract; however, incubation with column fractions eluted prior to the receptor gives an association rate constant similar to that for detergent extracts. The component responsible for this effect is nondialyzable and protease sensitive, indicating that it may be a protein or high-molecular-weight peptide. Affinity labeling experiments with [3H]propylbenzilylcholine mustard [N. J. M. Birdsall, A. S. V. Burgen, and E. C. Hulme (1979) Brit. J. Pharmacol. 66, 337-342] show radioactivity incorporated mainly in a broad peak of apparent molecular weight 75,000 +/- 5000.     

Proteoglycans from bovine nasal and articular cartilages. Fractionation of the link proteins by wheat germ agglutinin affinity chromatography

Two forms of link protein, 46 and 51 kDa, are present in proteoglycan aggregates from both bovine nasal and bovine articular cartilages. Studies reported here show that the link proteins bind to concanavalin A, Lens culinaris agglutinin, Ricinus communis agglutinin, soybean agglutinin, and wheat germ agglutinin lectins. When the link proteins are eluted from these lectins with appropriate competing sugars, the 46- and the 51-kDa link proteins elute together and no separation is achieved. However, when the link proteins bound to wheat germ agglutinin are eluted with a 0 to 4 M guanidine hydrochloride linear gradient, a good separation of the 46- and 51-kDa link proteins is achieved. Wheat germ agglutinin affinity chromatography has been used on a preparative scale to isolate the 51-kDa link protein from mature bovine articular cartilage to homogeneity, in amounts sufficient to examine its effect on proteoglycan aggregate size and stability in sedimentation velocity studies. Proteoglycan aggregates were reassembled from proteoglycan monomers and hyaluronate in the absence of link protein, in the presence of both 46- and 51-kDa link proteins, and in the presence of the individual 51-kDa link protein. The sizes of the aggregates were compared in terms of sedimentation coefficients (s(0)20). The stability of the aggregates was compared in terms of the per cent aggregate present at pH 7 and 5. At pH 7, the sedimentation coefficients (s(0)20) of link-free aggregates, aggregates formed with both link proteins, and aggregates formed with 51-kDa link protein were 72, 93, and 112 S, respectively. Thus, the 51-kDa link protein has a pronounced effect on aggregate size. The link-free aggregate was grossly unstable, and only 36% aggregate was present at pH 5. The aggregate formed with both link proteins was effectively stabilized against dissociation and 79% aggregate was present at pH 5. The aggregate formed with 51-kDa link protein was not effectively stabilized against dissociation, and only 60% aggregate was present at pH 5. Thus, despite its pronounced effect on aggregate size, the 51-kDa link protein does not effectively stabilize the proteoglycan aggregate against dissociation. These results suggest that the 51-kDa link protein may selectively increase aggregate size, while the 46-kDa link protein may be required to effectively stabilize the proteoglycan aggregate against dissociation.     

Single step recombinant human growth hormone (rhGH) purification from milk by peptide affinity chromatography

Recombinant human growth hormone (rhGH) is used for the treatment of several pathologies, most of them related to growth. Although different expression systems can be used for its production, the milk from transgenic cows is one of the most interesting due to the high rhGH level achieved (5 g/L). We have designed and synthesized short peptides (9 or 10 amino acid long) using Fmoc chemistry and studied their ability to purify rhGH from milk once immobilized on an agarose support. Using spiked milk with the hormone as a sample, rhGH was purified with 88% yield and 92% purity in a single step with a fold purification of 4.5. © 2018 American Institute of Chemical Engineers Biotechnol. Prog., 34:999–1005, 2018     

Suitability of immobilized metal affinity chromatography for protein purification from canola

This work demonstrates that proper selection of a metal ion and chelating ligand enables recovery of a his(6)-tagged protein from canola (Brassica napus) extracts by immobilized metal affinity chromatography (IMAC). When using Co(2+) with iminodiacetate (IDA) as the chelating ligand, beta-glucuronidase-his(6) (GUSH6) can be purified from canola protein extract with almost homogeneous purity in a single chromatographic step. The discrimination with which metal ions bound native canola proteins followed the order Cu(2+) < Ni(2+) < Zn(2+) < Co(2+) in regard to elimination of proteins coeluted with the fusion protein. IDA- and nitrilotriacetate (NTA)-immobilized metal ions showed different binding patterns, whose cause is attributed to a more rigid binding orientation of the his(6) in forming a tridentate with Me(2+)-IDA than in forming a bidentate with Me(2+)-NTA. The more flexible binding allows for multisite interactions over the protein.     

Serial lectin affinity chromatography with concanavalin A and wheat germ agglutinin demonstrates altered asparagine-linked sugar-chain structures of prostatic acid phosphatase in human prostate carcinoma

Differences between human prostate carcinoma (PCA, five cases) and benign prostatic hyperplasia (BPH, five cases) in asparagine-linked (Asn) sugar-chain structure of prostatic acid phosphatase (PAP) were investigated using lectin affinity chromatography with concanavalin A (Con A) and wheat germ agglutinin (WGA). PAP activities were significantly decreased in PCA-derived PAP, while no significant differences between the two PAP preparations were observed in the enzymatic properties (Michaelis–Menten value, optimal pH, thermal stability, and inhibition study). In these PAP preparations, all activities were found only in the fractions which bound strongly to the Con A column and were undetectable in the Con A unbound fractions and in the fractions which bound weakly to the Con A column. The relative amounts of PAP which bound strongly to the Con A column but passed through the WGA column, were significantly greater in BPH-derived PAP than in PCA-derived PAP. In contrast, the relative amounts of PAP which bound strongly to the Con A column and bound to the WGA column, were significantly greater in PCA-derived PAP than in BPH-derived PAP. The findings suggest that Asn-linked sugar-chain structures are altered during oncogenesis in human prostate and also suggest that studies of qualitative differences of sugar-chain structures of PAP might lead to a useful diagnostic tool for PCA.     

Large-scale purification of bovine brain lactate dehydrogenase by affinity chromatography on immobilized colchicine

Lactate dehydrogenase (LDH) [EC 1.1.1.27] in a crude extract (40-80% ammonium sulfate fraction) of bovine brain was adsorbed on an immobilized colchicine column and specifically eluted by addition of 1 mM NADH. The purity and subunit composition of the pooled LDH were estimated by two-dimensional gel electrophoresis. With an increase of NaCl concentration from 0 to 2.0 M, ligand saturation of LDH on immobilized colchicine increased from 6.8 to 14%, whereas that on immobilized Cibacron blue F3GA decreased from 2.1 to 0%. In the presence of high NaCl concentration, immobilized colchicine enabled both large- and small-scale purification of LDH by affinity chromatography and resulted in a yield of 117 mg from 1 kg of bovine brain in the presence of 2.5 M NaCl or higher recoveries of 54-96% from various tissues of one rat in the presence of 1.0 M NaCl. These results indicate that immobilized colchicine is an excellent adsorbent for the isolation and purification of LDH by affinity chromatography and has a high LDH-adsorbing capacity dependent upon a high NaCl concentration. Kinetic studies revealed that colchicine apparently competed with cofactor NAD for the active site of LDH and the Ki values of colchicine decreased with an increase of NaCl concentration. The chemical specificity of the colchicine-binding site of LDH was studied by the use of colchicine analogues and it is concluded that both the tropolone moiety (C-ring) and the amido bond in a side chain of colchicine structure are essential to the colchicine-LDH interaction.     

Rapid purification of annexin V from human placenta by affinity chromatography

Affinity purification of annexin V from human placenta on column with appropriate monospecific antibodies is developed. The procedure permits purification of the protein to a highly purified state by a two stage procedure. The yield of the protein is about 5 mg per 100 g of wet tissue. Because of high homologies between various annexins, it was supposed that this procedure can be also applied for purification of other annexins from other tissues.     

The affinity of Concanavalin A and wheat germ agglutinin for components of the muscle spindle

Summary Sections through the soleus muscle of the rat were incubated with concanavalin A (Con A) or wheat germ agglutinin (WGA) conjugated to fluorescein isothiocyanate. Binding of these lectins to structures which comprise the muscle spindle was studied by fluorescence microscopy. The distribution of the lectins was heaviest in the outer capsule of the spindle and at the surface of intrafusal muscle fibres. The periaxial space in the equatorial region of spindles was unlabelled except in the immediate vicinity of the axial bundle. Binding by Con A was more extensive than by WGA, suggesting that more glucopyranoside units are accessible within the muscle spindle than are those of N-acetylglucosamine.     

Stimulation and inhibition of human T cell subsets by wheat germ agglutinin

Wheat germ agglutinin (WGA), a tetravalent lectin, has both stimulatory and inhibitory effects on human T lymphocytes. It has been suggested that these actions are related and that WGA selectively stimulates a suppressive subset of T cells. We studied the ability of WGA to stimulate and inhibit subpopulations of human peripheral blood mononuclear cells (PBMC) known to have helper or suppressor activity. Fresh human PBMC were depleted of either T4+ or T8+ cells by using antibody-mediated complement lysis. The resultant cell populations were stimulated with WGA, and the proliferative response was assessed by [3H]thymidine incorporation, IL 2 receptor expression, the ability to elaborate IL 2 in culture supernatants, and the susceptibility to inhibition by the monoclonal antibody anti-Tac. Similar experiments with cells from a WGA-responsive continuous T cell culture were also performed. WGA inhibited phytohemagglutinin (PHA)-induced proliferation of PBMC depleted of either T4+ or T8+ cells. WGA also inhibited PBMC that had been depleted of adherent cells and Ia+ cells and then induced to proliferate with a combination of TPA and PHA. Our findings indicate that WGA induces IL 2-dependent proliferation in a small proportion of both T4+ and T8+ lymphocytes. We also provide evidence that the inhibitory activity of WGA is not mediated by a T4+, T8+, or Ia+ cell, suggesting that WGA acts directly on the proliferating cell rather than selectively stimulating a suppressive subpopulation.     

The platelet activation induced by wheat germ agglutinin

In human platelets, wheat germ agglutinin (WGA) induced serotonin release without cell agglutination. WGA induced the phosphorylation of both 40-kDa and 20-kDa proteins in a parallel manner, and at least, the phosphorylation of 40-kDa protein was preceded by transient formation of endogenous diacylglycerol (DG) accompanied by a decrease in phosphatidylinositol (PI). Both phosphorylation of these two proteins and serotonin release were inhibited by prior treatment of platelets with dibutyryl cyclic AMP, W-7, or TMB-8. These results suggest that both phosphatidylinositol turnover and Ca2+ mobilization play an essential role in WGA-induced platelet activation.