Isolation of membrane glycoproteins by affinity chromatography in the presence of detergents.

Wheat germ agglutinin has been used in a one-step preparative method to isolate the major sialoglycoprotein (glycophorin A) from the human erythrocyte membrane. The conditions for isolation and purification of the sialoglycopeptide included low concentration of sodium dodecyl sulfate in the presence of relatively high salt concentration. This medium caused complete solubilization of the membrane but still allowed almost quantitative binding of the sialoglycopeptide to wheat germ agglutinin-Sepharose. The eluted protein from such affinity systems was found to be chemically comparable to glycophorin A, as prepared by other procedures.     

Isolation of the membrane glycoproteins of human blood platelets by lectin affinity chromatography

The major platelet membrane glycoproteins have been solubilized in 1.0% sodium deoxycholate and subjected to affinity chromatography on the lectins from Lens culinaris, wheat germ and Abrus precatorius. Polyacrylamide gel electrophoresis in the presence and absence of a reducing agent together with the differential binding of the lectins to the glycoproteins permitted the distinction of at least seven separate glycoprotein entities. A new nomenclature for the glycoproteins is proposed to accomodate the additional data.Using combinations of lectin columns, glycoproteins Ia and Ib could be prepared in a pure state and IIb and IIIa could be greatly purified. The binding of lectins to glycoprotein Ib has been strongly implicated as a necessary step in the aggregation response of platelets to lectins.     

Isolation of ADP-ribosyltransferase by affinity chromatography

An affinity adsorbent for ADP-ribosyltransferase (EC 2.4.2.30) has been synthesized by coupling 3-aminobenzamide to Sepharose 4B. Using this material, ADP-ribosyltransferase from human placenta has been purified from crude extract to homogeneity within a few hours. The enzyme has an apparent Km for NAD+ of 52 microM. Its molecular mass is 115,000 as determined by gel electrophoresis. The enzyme is DNA dependent and stimulated by histone, its temperature optimum is at 25 degrees C, and its pH optimum is around pH 9. alpha-NAD+, thymidine, caffeine, theophylline, theobromine, 3-methoxybenzamide, and nicotinamide inhibit the enzyme. Purification of ADP-ribosyltransferases from horse, rat, and chicken liver was also achieved with the method described.     

Isolation of fucosyl glycoproteins from human erythrocyte membranes by affinity chromatography using Aleuria aurantia lectin

Fucosyl glycoproteins were fractionated from a sialoglycoprotein preparation of human erythrocyte membrane by using Aleuria aurantia lectin (AAL) coupled to Sepharose 4B. The affinity eluates were characterized as having high fucose content and significant H activity as measured in terms of N-acetylgalactosamine (GalNAc) incorporation with A1-enzyme and hemagglutination inhibition assay with anti-H sera, and the unadsorbed fractions contained low levels of fucose and were devoid of apparent H activity. Neuraminidase treatment of the material improved the recovery of the affinity eluate. Thus, 66% of the applied asialoglycoprotein was recovered in the eluate, though only 10% of the untreated material was bound and eluted. Moreover, a fucose-rich and H-active fraction was obtained through the affinity chromatography of the previously unbound fraction after neuraminidase treatment. In sodium dodecyl sulfate-gel electrophoresis, the main component of both the unadsorbed and eluted fraction was revealed to be PAS-1 glycoprotein. These results indicate that AAL-Sepharose was effective for isolating fucose-containing compounds from the membrane glycoprotein especially after neuraminidase treatment. The reasons for the appearance of H activity in the affinity eluates are discussed.     

Isolation by affinity chromatography of specialized membrane fractions from cat liver microsomes]

Microsomal glucokinase is solubilized by incubation in the presence of several metabolites. After solubilization of the enzymes, the membranes present free sites for specific binding of glucokinase, therefore, they can be purified by affinity chromatography on Sepharose--ATP-glucokinase. This method yields membranous vesicles which contain, in addition to glucokinase, uridylyl-transferase, phosphoglucomutase, sialyl-transferase and adenylate cyclase. Galactosyl-transferase, glucose-6-phosphatase and NADPH cytochrome c reductase are absent. It appears that functionally related enzyme from UDP-glucose biosynthesis are aggregated onto specific patches of the membrane, most likely from Golgi apparatus.     

Isolation of brain tubulin by affinity chromatography

An affinity chromatographic procedure for the isolation of tubulin from brain is described. The yield is good and the method rapid and gentle. Criteria of purity are colchicine binding, SDS acrylamide gel electrophoresis, and velocity sedimentation.     

Isolation of phosphoglycerate kinases by affinity chromatography

A variety of Sepharose derivatives containing DL-O-phosphorylserine or adenosine nucleotides with different points of attachment, has been synthesized and tested for affinity to phosphoglycerate kinase. The most effective gels contained periodate-oxidized ATP or ADP bound via the ribose by hydrazone formation to adipoyl-dihydrazo-Sepharose. The effect of pH, magnesium and buffer ions on the binding capacity of the ATP derivative of Sepharose has been examined. Optimal elution of phosphoglycerate kinase was investigated using different combinations of adenosine nucleotides, 3-phosphogylcerate and magnesium ions. A method is presented giving conditions for the purification of phosphoglycerate kinase from different sources (spinach, human erythrocytes, human, rabbit and trout muscle). It includes extract preparation, affinity chromatography and gel filtration. The method is greatly superior to known isolation procedures by virtue of its technical simplicity, excellent yield (85-100%) and reproducability. The capacity of the ATP-ribosyl-adipoyl-dihydrazo-Sepharose was 5 mg phosphoglycerate kinase per 1 g of matrix. Polyacrylamide gel electrophoresis in the presence of sodium dodecylsulfate indicated that the final products are homogeneous. The phosphoglycerate kinases from different sources appear to have the same affinity for this ATP derivative of Sepharose, the same molecular weight and the same specific activity.     

Isolation of tyrosinase-mRNA by affinity chromatography of polysomes

We present a method whereby some mRNAs which code for enzymes can be isolated by affinity chromatography of newly synthesized polypeptides bound to their polysome complexes. Using this method we have isolated tyrosinase-mRNA from Xenopus laevis oocytes and have analysed the translation products from the RNAs thus obtained. In vitro translation reveals the presence of two mRNAs coding for polypeptides of molecular weights of 20 000 and 32 000, respectively. The larger molecule corresponds to the molecular weight of nascent tyrosinase. Furthermore, microinjection of the mRNA into Xenopus oocytes results in the synthesis of active tyrosinase. Since this isolation method is dependent on the ability of nascent enzymes to bind to their substrate analogue, it is thought that this approach may be appropriate for obtaining mRNAs coding for other enzymes.     

Isolation of cathepsins D by affinity chromatography]

The paper deals with the isolation of cathepsin D from rat liver, chicken liver and bovine spleen by affinity chromatography. The synthesis of the adsorbent was performed using the competitive inhibitor of cathepsins D and pepsin pepstatin and activated Sepharose. Application of a pepstatin-Sepharose column allows obtaining a highly active and purified enzyme in a short period of time.     

Isolation of lipid-free C-reactive protein by affinity chromatography

Human C-reactive protein purification has been hampered by its association with lipids. Isolation of pure lipid-free C-reactive protein was obtained by a three step procedure. First, partially lipid-free C-reactive protein was obtained by affinity chromatography from ascitic fluids; second, lipid-bound proteins were eliminated by calcium-dependent precipitation; and third, lipid-free pure C-reactive protein was obtained by affinity re-chromatography of the supernatant. A 46-50% yield of lipid-free C-reactive protein was obtained compared with the 14.7% obtained by the old method of extraction with lipid solvents.     

Isolation of antigens and antibodies by affinity chromatography

Antibody-antigen binding constants are commonly strong enough for an effective affinity purification of antibodies (by immobilized antigens) or antigens (by immobilized antibodies) to work out a straightforward purification method. A drawback is that antibodies are large protein molecules and subject to denaturation under conditions required for the elution from the complex. Structures of antigens can vary but usually antigens are also equally subject to similar problems. The lability of the components can sometimes make the procedure sophisticated, but usually in all cases it is possible to find a satisfactory approach. In certain cases, specific interactions of the Fc part of antibodies are more facile to exploit for their purification.     

Isolation of bovine seminal ribonuclease by affinity chromatography

Pyrimidine base-specific RNase was isolated from bull semen by ammonium sulfate precipitation followed by affinity chromatography with ATP-ribosyl-adipoyldihydrazo-Sepharose. The enzyme is also bound to the AMP- or CMP-Sepharose gel. The binding capacity is 7 mg RNase/ml ATP-Sepharose. Using this procedure, a homogeneous protein with 74% yield could be isolated. The enzyme is a dimer with a molecular weight of 26,000.     

Isolation of chromosaponin I-specific antibody by affinity chromatography

Chromosaponin I (CSI), a gamma-pyronyl-triterpenoid saponin isolated from pea and other leguminous plants, modulates several developmental processes of plant roots and activates the sugar taste receptor cells in blowflies. CSI is a unique saponin for its reducing power and biological activities in both plants and insects. In the present paper, we described the method of preparation for CSI-specific antibody using CSI-affinity and soyasaponin I-affinity columns. The antibody's-specific binding activity to CSI was confirmed by a bioassay using Arabidopsis roots and a ligand-molecule interaction analysis using BIAcore 3000. Because of the lability of CSI, the CSI-affinity column was made only by a moderate reaction condition in which CSI was coupled to EAH Sepharose 4B in the presence of 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide hydrochloride (EDC). The special control of the reaction temperature was essential to complete the coupling reaction; the reaction with EDC at 0 degrees C followed by a gradual increase in temperature.     

Isolation of soybean lipoxygenase-2 by affinity chromatography

Isoenzyme lipoxygenase-2 from soybean was isolated by affinity chromatography. Gel electrophoresis showed it to be a single protein. Its pH optimum of 6.5, range of 5.0–8.0 and activity which increased when Ca²⁺ was added identified the isolated enzyme as lipoxygenase-2.     

Immobilized metal affinity chromatography in the presence of arginine

Arginine hydrochloride (ArgHCl) is a versatile solvent additive, as it suppresses protein aggregation. ArgHCl has been used for protein refolding and to solubilize proteins from loose inclusion bodies. Immobilized metal affinity chromatography (IMAC) is one of the most commonly used technologies for purification of recombinant proteins. Here we have evaluated compatibility of ArgHCl with IMAC purification for his-tag proteins. ArgHCl clearly interfered with protein binding to Ni-columns. Nevertheless, such interference was greatly reduced at ArgHCl concentration below 200 mM, demonstrating that IMAC purification can be done even in the presence of ArgHCl.