Isolation and purification of biopolymers using an affinity chromatography method. IX. Preparation, properties and use of affinity adsorbents with immobilized polypeptide fragments of collagen

Synthesis and properties of new affinity adsorbents with immobilized polypeptide fragments of collagen molecule (alpha-chains, beta-components, cyanogen bromide peptides) were described. Adsorbents with alpha-chains and alpha 1CB7-peptide had fibronectin binding capacity 1.5-2.0 times higher than commercial gelatin-Sepharose. Commercial production of highly purified fibronectin from human plasma using affinity chromatography on immobilized individual alpha-chains of collagen was developed.     

Isolation and purification of angiotensin II using affinity and high-pressure liquid chromatography

Peptides have been found in a variety of tissues including brain. To purify the peptide angiotensin II, a three-step method for the isolation and purification has been developed using extraction, affinity chromatography, and high-pressure liquid chromatography. Angiotensin II antiserum purified by affinity chromatography was covalently coupled to Affi-gel 10 (Affi-gel 10-AB). The efficiency and usefulness of this column for the purification of angiotensin II from biological sources were tested with 125I- and 3H-labeled (Ile5)-angiotensin II added to rat brains prior to extraction. After extraction, the recoveries for both peptides were 74 and 75%, respectively. Recovery after the purification on Affi-gel 10-AB was 84 and 82%. Thirty-two percent of the radioactivity was not retained and 50% of the radioactivity could be eluted with 0.1 M Na citrate buffer containing 1 M NaCl using a stepwise pH gradient. Characterization by HPLC of the unretained radioactivity from the Affi-gel 10-AB column showed one peak for [125I]angiotensin II, coeluting with the [125I]angiotensin II standard and two minor peaks. Only 30% of unretained [3H]angiotensin II could be identified as intact [3H]angiotensin II on HPLC. Both [125I]angiotensin II and [3H]angiotensin II elutable at pH 5.0 and 4.0 on Affi-gel 10-AB could be demonstrated as highly purified [125I]angiotensin II and [3H]angiotensin II on HPLC with a purity of more than 90%. On HPLC, the recovery was 81% for [125I]angiotensin II and 99% for [3H]angiotensin II. The recovery for the entire three-step procedure was about 60%. The loading capacity of the Affi-gel 10-AB column for (Ile5)-angiotensin II was 550 ng.(ABSTRACT TRUNCATED AT 250 WORDS)     

Isolation and purification of cytokinin binding protein from tobacco leaves by affinity column chromatography

Cytokinin binding protein from tobacco leaves was isolated and purified to a single protein by means of affinity chromatography on benzyladenine-linked Sepharose column combined with polyacrylamide gel electrophoresis. In vitro binding of this protein to [¹⁴C] benzyladenine was inhibited remarkably by cold benzyladenine and kinetin and slightly by adenine, but not adenosine. The molecular weight of the protein was determined to be about 4,000 daltons by gel filtration and SDS polyacrylamide gel electrophoresis.     

Isolation and purification of morphine receptor by affinity chromatography

Brain membranes were solubilized by sonication and Triton X-100 extraction and applied to an affinity column consisting of a 6-succinyl morphine derivative of Affi Gel-102. A fraction exhibiting high opiate binding was eluted by tris-buffer containing naloxone, CHAPS and NaCl. This fraction consisted of both proteins and acidic lipids. The opiate binding properties of this purified material exhibited many properties similar to those of membrane bound receptors of the u-type, including high affinity, stereospecificity, Na-effect and rank order in affinity for opiates. This opiate binding material was highly sensitive to both trypsin and N-ethylmaleimide. Based on the protein content of the isolated membrane receptor, a 3200-fold purification over the original brain P2 fraction was achieved.     

Isolation of human lactate dehydrogenase isoenzyme X by affinity chromatography.

Human isoenzyme LDH-X (lactate dehydrogenase isoenzyme X) was isolated from seminal fluid of frozen semen samples by affinity chromatography by using oxamate-Sepharose and AMP-Sepharose. In the presence of 1.6 mM-NAD+, isoenzyme LDH-X does not bind to AMP-Sepharose, whereas the other lactate dehydrogenase isoenzymes do. This is the crucial point in the isolation of isoenzyme LDH-X from the other isoenzymes. The purified human isoenzyme LDH-X had a specific activity of 146 units/mg of protein.     

Synthesis of a biospecific adsorbent for the purification of the three human retinoic acid receptors by affinity chromatography.

The total synthesis of an affinity gel suitable for the purification of retinoic acid receptors (hRARs) is reported. A chalcone derived from a potent retinobenzoic acid (Ch55) was chosen as the ligand and fixed to an immobilized matrix by coupling with the N-hydroxysuccinimide ester of agarose (Affi-Gel 10, Bio-Rad Laboratories). Efficiencies of purification of the different human RARs were tested, using recombinant receptors produced with the baculovirus expression system.     

Progesterone binding components of chick oviduct. X. Purification by affinity chromatography.

Cytoplasmic progesterone receptors of chick oviduct have been purified in 8% yield by steroid affinity and ion exchange chromatography. The affinity resin, deoxycorticosterone-bovine serum albumin-Sepharose, binds progesterone receptors with high affinity (KD equals 8 times 10-minus 10 M) and its use resulted in a greater than 2000-fold purification over the starting material in a single step. DEAE-Sephadex A-50 chromatography was then used to achieve final purification. NA dodecyl-SO4 gel electrophoresis and DEAE-cellulose chromatography showed that the purified receptors contained both of the previously described 4 S progesterone binding components in near equal amounts. Na dodocyl-SO4 gel electrophoresis also showed that these components consisted of single polypeptide chains with molecular weights of 110, 000 (A component) and 117, 000 (B component). There was no evidence for subunits of lower molecular weight. The purified materials have identical hormone-binding kinetics and steroid specificity to crude cytosol receptors. The isolated receptors retain the three biologically important properties exhibited by progesterone binding components present in cruder preparations: they bind specifically to (a) nuclei (KD equals 1.1 times 10-minus 9 M, 10, 000 sites per nucleus); (b) chromatin (KD equals 3 times 10-minus 9 M, 2000 sites per pg of DNA-);and (C) DNA.     

Separation of fibronectin from a plasma gelatinase using immobilized metal affinity chromatography.

Conventional preparations of plasma fibronectin are known to contain a co-purifying gelatinase [1986, J. Biol. Chem. 261, 4363-4366], but so far useful methods to remove the protease have not been available. In this study a number of different methods were tested in order to achieve separation of the two proteins. Immobilized metal affinity chromatography was found to be efficient for this purpose, and a convenient procedure to separate the two proteins under nondenaturing conditions on chelating Sepharose charged with Co2+, Ni2+, or Zn2+ is described. An alternative method employing pH gradient elution of an Fe3+ gel also resolved fibronectin from the gelatinase. The Fe3+ gel bound both proteins at pH 6.0 but not at pH 7.4, suggesting that the two proteins were phosphorylated. The described procedures will now allow studies of the functions of fibronectin in the absence of the contaminating protease.     

The purification of cytochrome f and plastocyanin using affinity chromatography

Both plastocyanin and cytochrome f were purified using a combination of affinity chromatography together with established methods. Plastocyanin was partially purified using the method of Davis and San Pietro (Anal. Biochem. 95 (1979) 254-259), after which it was further purified using a column of cytochrome c covalently attached to Sepharose 4B. The affinity column was prepared using the method of Godinot and Gautheron (Methods Enzymol. 54 (1979) 112-114). The final purity index ratio (A278/A597) was less than 1.2, which is equal to that obtained using the more expensive FPLC procedure (Anderson, G.P., Sanderson, D.G., Lee, C.H., Durell, S., Anderson, L.B. and Gross, E.L. (1987) Biochim. Biophys. Acta 894, issue 3). Cytochrome f was partially purified using a modification of the method of Matazaki et al. (Plant Cell. Physiol. 16 (1975) 237-246) and bound to an affinity column of plastocyanin covalently attached to Sepharose 4B. Cytochrome f purified using this procedure had a purity index ratio (A554.5/A277) of 1.2. Both proteins are tyrosine proteins containing no tryptophan residues. After the affinity chromatography step, the fluorescence emission spectrum of either plastocyanin or cytochrome f was typical of a tyrosine protein free from tryptophan contamination.     

Structure and affinity of DNA binding peptides.

Artificial peptides designed to form alpha-helical, beta-turn, antiparallel beta-sheet and beta-hairpin structures which are among the motifs most frequently found in natural DNA/RNA binding proteins were synthesized and their characteristic features were examined in the presence or absence of double or triple stranded DNA by means of UV melting experiments, CD spectra, SPR measurements. It was revealed that amphiphilic character arising from the specific secondary structures and positive charge in the hydrophobic face of peptides played an important role in the interaction with DNA, and that hybrid duplex and triplex were intensively stabilized by the cationic amphiphilic peptides. It was also found that these peptides could protect dsDNA against DNase 1 digestion. These results indicate that structurally designed amphiphilic peptides synthesized in the present study can be powerful tools for antisense and antigene strategies.     

Isolation of human proteasomes and putative proteasome-interacting proteins using a novel affinity chromatography method

The proteasome is the primary subcellular organelle responsible for protein degradation. It is a dynamic assemblage of 34 core subunits and many differentially expressed, transiently interacting, modulatory proteins. This paper describes a novel affinity chromatography method for the purification of functional human holoproteasome complexes using mild conditions. Human proteasomes purified by this simple procedure maintained the ability to proteolytically process synthetic peptide substrates and degrade ubiquitinated parkin. Furthermore, the entire purification fraction was analyzed by mass spectrometry in order to identify proteasomal proteins and putative proteasome-interacting proteins. The mild purification conditions maintained transient physical interactions between holoproteasomes and a number of known modulatory proteins. In addition, several classes of putative interacting proteins co-purified with the proteasomes, including proteins with a role in the ubiquitin proteasome system for protein degradation or DNA repair. These results demonstrate the efficacy of using this affinity purification strategy for isolating functional human proteasomes and identifying proteins that may physically interact with human proteasomes.     

Purification of human plasma fibronectin using immobilized gelatin and Arg affinity chromatography

This protocol describes a method for purification of fibronectin (Fn) from human plasma based on a combination of gel filtration and affinity chromatography steps. Clarified plasma is first loaded onto a Sepharose CL-4B column and unbound material is sequentially purified on columns containing covalently coupled gelatin and Arg. The elution conditions are optimized to obtain a homogeneous preparation of Fn on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Although the Fn yield is expected to be lower than that obtained using other methods, affinity adsorbents based on gelatin and Arg and gentle elution steps offer advantages including a high purity of the preparation and a correctly folded protein. The preparation can be useful for interaction studies and analysis of biological and immunological activities of Fn.     

A method for the purification of histone fraction F3 by affinity chromatography

Calf thymus histone fraction F3 has been purified in high yield by affinity chromatography. The protein is attached reversibly to an organomercurial—Sepharose—by the thiol groups of its cysteinyl residues, while all other histones lacking -SH groups pass through the column. Histone F3 is subsequently eluted with -SH reagents of low molecular weight, such as cysteine or β-mercaptoethanol. The purified F3 shows electrophoretic heterogeneity due to different degrees of acetylation of the parent polypeptide chain. Under oxidizing conditions it forms a complex mixture of dimers and oligomers, all of which have some available -SH groups.     

Isolation, purification and biochemical characterization of human placental interferons by tandem high-performance affinity chromatography.

Human placental trophoblasts, fibroblasts and the trophoblast-derived malignant cell JAR are potent producers of interferons (IFNs) when stimulated with Sendai virus. The three cell lines produced different levels and compositions of IFN-alpha subtypes and IFN-beta. Anti-IFN globulins, Cibacron Blue F3GA and Concanavalin A were covalently immobilized on pressure-stable, macroporous polymeric matrices derivatized with vinyl sulphone (HEMA-BIO 1000 VS and HEMA 1000 VS). These supports were packed in biocompatible PEEK columns and were coupled with switching valves, to develop a tandem high-performance affinity chromatographic (HPAC) method for the isolation, purification and biochemical characterization of the IFNs produced in Sendai virus-stimulated human placental trophoblasts, fibroblasts and trophoblast-derived malignant cell, JAR, cultures. Silver-stained SDS-PAGE and gel densitometric analysis revealed the purity of the purified proteins to be between 94 and 98%. Specific activities of the purified IFNs ranged between 0.37-2.76 x 10(8) IU/mg of protein with cumulative recoveries between 90 and 92.2%. The purified IFN components exhibited quantitatively different antiviral activities in human and bovine cell lines. The utility of the tandem method for the purification and characterization of human type 1 IFNs produced from other cell lines are also discussed.     

Isolation and purification of phospholipase C from Clostridium perfringens and antibodies against it using polymeric biospecific adsorbents

A new polymeric biospecific adsorbent intended for isolation of Clostridium perfringens phospholipase C (PLC) and PLC-specific antibodies is discussed. It was obtained by radical copolymerization of acrylamide, methylenbisacrylamide, and the acryl acid chloranhydride-acylated substrate of PLC (chicken yolk lecithovitellin). Maximal adsorption of PLC was observed in the presence of the enzyme activator, and the highest amount of PLC was eluted in case of its minimal adsorption. The "residual" activity of PLC on the adsorbent was used to isolate homologous antibodies from the anti-perfringens serum. PLC and PLC-specific antibodies were serologically pure in the agar precipitation test with the anti-perfringens serum and the primary PLC concentrate, respectively; the antibodies gave only one zone in PAAG-electrophoresis.     

Partial purification of guinea pig MIF by affinity column chromatography using macrophages.

First we have confirmed the previous observation that the macrophage migration inhibitory factor (MIF) was adsorbed on normal peritoneal macrophages when they were incubated at 4 C for 60 min. It was found that macrophages fixed with 2% glutaraldehyde gave more reproducible results than viable cells in terms of "adsorption" of guinea pig MIF. The adsorption was achieved more completely at 37 C than at 4 C, indicating that this reaction is a temperature-dependent phenomenon. Using these glutaraldehyde-fixed macrophages, a kind of cell-affinity column was successfully developed. The guinea pig MIF preparation lost its activity when it was passed through this affinity column, and MIF adsorbed on the column was recovered by elution with 0.1 M (L)-fucose of 0.1 M (D)-glucose. Such MIF active eluate was found to be at least 30--40 fold more pure than the original MIF preparation which had been previously fractionated according to its molecular weight. Therefore, this type of macrophage-affinity column may be useful for the purification of MIF.