Affinity chromatography system for parallel purification of recombinant protein samples

In protein engineering, the tasks of generating and testing a large number of variants of a molecule and of optimizing expression conditions for one distinct molecule create the need for purification methods that can handle a large number of samples simultaneously. We describe the development and some application results of a simple affinity chromatography system that can be used for the parallel purification of 24 protein samples, yielding sufficient quantities for biochemical and functional analysis. Advantages of this system over existing systems are as follows. Compared with commercially available complete chromatography systems, the costs of this system are minimal. In comparison with vacuum systems with various outlets, and with batch purification systems where centrifugation is necessary, this system allows gentler processing of the samples. This could be important for proteins that are easily damaged.     

Affinity chromatography of lipoxygenases.

A number of aminohexyl agarose derivatives of unsaturated fatty acids have been prepared and evaluated as materials for the affinity chromatography of soybean and pea lipoxygenases. A practical method for a one-stage purification of soybean lipoxygenase-1, with a purification factor of 16, is described, using either linolenate or docosa-4,7,10,13,16,19-hexaenoate as ligands. Results show that alleged competitive inhibitors do not cause sharp elution from the affinity column, and that there is an increasing specificity of binding and sharpness of elution as the proportion of unsaturation in the ligand is increased. These results are discussed in terms of the relative importance of the types of bonding involved in enzyme-substrate binding.     

Affinity chromatography of phosphofructokinase.

The behavior of mammalian phosphofructokinase on immobilized adenine nucleotides was investigated. Three different insolubilized ligands were compared using a pure rabbit muscle phosphofructokinase. N⁶-[(6-aminohexyl)-carbamoyl-methyl]-ATP-Sepharose bound at least 90 times more enzyme than either N⁶-(6-aminohexyl)-AMP-agarose or ATP-adipic acid hydrazide-Sepharose. The elution of phosphofructokinase from the ATP-Sepharose with various metabolites and combinations of metabolites was investigated. The enzyme is eluted specifically from N⁶-[(6-aminohexyl)-carbamoyl]-ATP-Sepharose with a mixture of 25 μm each of fructose 6-phosphate and ADP (±Mg²⁺). The enzyme is not eluted either with ATP (25 μm), fructose 1,6-diphosphate (1 mm), ADP (25 μm), fructose 6-phosphate (1 mm) alone, or with a mixture of fructose 1,6-diphosphate (25 μm) and ATP (25 μm). The recovery of bound enzyme was usually greater than 90%. A mixture of glucose 6-phosphate and ADP or a mixture of IDP and fructose 6-phosphate also elutes the enzyme, but the recovery with these eluants was only about 40%. It was concluded that the “dead-end” complex is the most effective in the elution. Using this method, phosphofructokinase has been prepared in an essentially homogeneous form from muscle and brain of rabbit and rat. The overall isolation procedure involves a high speed centrifugation of crude extracts which sediments phosphofructokinase as a pellet, followed with adsorption on N⁶-[(6-aminohexyl)-carbamoyl-methyl]-ATP-Sepharose and specific elution with the mixture of fructose 6-phosphate and ADP.     

Affinity chromatography on immobilized hog gastric mucin and ovomucoid. A general method for isolation of lectins

Two affinity adsorbents of general applicability for isolating of plant lectins are presented. The isolation of 30 lectins from 27 plants is described. The method works quickly and is inexpensive. In particular, mixtures of different lectins occurring in some plants may be resolved in one run.     

Affinity chromatography of human anti-dextran antibodies isolation of two distinct populations

Affinity chromatography is a very efficient method for antibody purification. Two affinity chromatography supports were prepared to analyze the specificity of anti-dextran antibodies. Silica beads were grafted with native dextran or with functionalized dextran. The anti-dextran antibodies present in some human sera were analyzed by enzyme-linked immunosorbent assay method. These antibodies play an important role in severe dextran-induced anaphylactic reactions in humans by forming immune complexes with clinical dextran. The results indicated that two distinct populations of anti-dextran antibodies were purified from human serum, using dextran-coated silica beads. Elution from this support with an oligo-dextran of 4000 g/mol allowed the isolation of one population that only recognized native dextran as antigen. Functionalized dextran coated on dextran silica beads led to the purification, with a glycine-HCl buffer, of another subclass of antibodies that recognized substituted dextran derivatives. Furthermore, these antibodies could be useful tools for in vitro and in vivo investigations using dextran derivatives as bio-active polysaccharides.     

Affinity chromatography reveals RuBisCO as an ecdysteroid-binding protein

The aim of this work was to isolate plant ecdysteroid-binding proteins using affinity chromatography. Ecdysteroids as insect hormones have been investigated thoroughly but their function and the mechanism of action in plants and other organisms is still unknown although ecdysteroids occur in some plants in a relatively large amount. Therefore, 20-hydroxyecdysone was immobilized on a polymeric carrier as a ligand for affinity chromatography in order to isolate plant ecdysteroid-binding proteins from the cytosolic extract of New Zealand spinach (Tetragonia tetragonoides). Non-specifically bound proteins were eluted with a rising gradient of concentration of sodium chloride, and 3% (v/v) acetic acid was used for the elution of the specifically bound proteins. Using this method, ribulose 1,5-bisphosphate carboxylase/oxygenase (RuBisCO) was isolated. The influence of ecdysteroids on RuBisCO was further studied. Our results show that ecdysteroids are able to increase the yield of RuBisCO-mediated reaction in which CO(2) is fixed into organic matter by more than 10%.     

Affinity chromatography of cysteine-containing histone.

Affinity chromatography based on the reaction between SH groups in protein and +HgC6H4CO groups in the p-mercuribenzoylaminoethyl derivative of Sepharose 4B was examined with a crude preparation of calf thymus cysteine-containing histone. Adsorption of the histone onto the column by specific coupling was found to be optimal in 0.1 M citrate buffer, pH 5.5, containing 5M urea to prevent any aggregation of histones and their non-specific adsorption onto the column, and elution from the column was successfully performed by cleavage of the resulting S-Hg bond with urea-buffer solution containing 0.05 M 2-mercaptoethanol. Under these conditions both the adsorption and elution were quantitative; no adsorption was observed when either SH-blocked histone or unsubstituted Sepharose was used. The cysteine-containing histone thus recovered, after further purification by Bio-Gel P-60 chromatography to remove some cysteine-containing nonhistone proteins contaminating the starting material, showed a single band on polyacrylamide gel electrophoresis and an amino acid composition agreeing with the known sequence of this histone.     

Affinity chromatography of tyrosine transfer RNAs.

Ricinus communis agglutinin, a lectin from castor beans has an affinity for β-d-galactose and tyrosine tRNAs of mammalian tissues have galactose in gal-Q base of their anticodons. We have studied interaction between tyrosine tRNAs and this lectin immobilized on solid supports using spacer arms of different lengths. Tyrosine tRNAs are separated from nineteen other tRNAs of bovine liver by affinity chromatography using the lectin immobilized to an agarose matrix. The results indicate that a spacer arm length of 10 Å between the agarose bead and the lectin gives the best separation. Two tyrosine tRNA isoacceptors are separated from each other and from other tRNAs in one step using this affinity column chromatography.     

Affinity chromatography of pig heart fumarase.

2-(5'-Phenylpentyl)fumaric acid was shown to be a competitive inhibitor (Ki 0.5 mM) of pig heart fumarase. After nitration of the aromatic ring, reduction to the amine and diazotization, the acid was attached via azo linkages to a Sepharose 4B-tyramine matrix. The resulting adsorbent was used for the affinity chromatography of crude fumarase, purifications of approx. 20-fold being obtained by specific elution with 0.01 M-citrate.     

Affinity chromatography of the phosphatidylcholine exchange protein from bovine liver

Affinity chromatography has been used to purify the phosphatidylcholine exchange protein from bovine liver. The affinity resin consisted of 1-acyl-2-(9-carboxy)nonyl-glycero-3-phosphocholine linked to AH-Sepharose 4 B via the carboxyl group. Application of a crude exchange protein fraction to the affinity column resulted in a complete adsorption of the phosphatidylcholine exchange protein. The exchange protein eluted with a buffer containing 0.15% sodium deoxycholate. The most active fraction was 130-fold purified and accounted for 62% of the activity.     

Affinity chromatography of transaminases

It is shown that glutamic-oxaloacetic transaminase from pig heart can be selectively and reversibly bound to a Sepharose column substituted with N′-alkyl derivatives of pyridoxamine 5′-phosphate. A simple procedure is described which includes formation of apotransaminase, its binding to the substituted Sepharose, and its elution under the same conditions that were used to prepare the apoenzyme. There is no binding of the transaminase to Sepharose substituted with unmodified pyridoxamine 5′-phosphate, indicating the importance of a certain “length” of the substituent for binding.     

Affinity chromatography of aminopeptidases

The recent data are generalized concerning a series of synthetic oligopeptides which are competitive inhibitors of aminopeptidases of animal, plant and microbic origin. A method for biospecific chromatography of these enzymes is developed, using as ligands such inhibitors as diazo derivatives of p-aminophenyl-, chloromethyl- and methylketones of L-amino acids and peptides, amino acids, aliphatic acid amides. It is established that the most effective inhibitors of aminopeptidases contain L-amino group in the uncharged form in the N-end position, hydrophobic lateral chain of L-configuration and a carbonyl group analogous to position of these groups in the substrate. Methods for synthesis of certain peptides are developed with respect to the above requirements. It is shown that peptides with a space-inaccessible peptide link and antibiotics are often used as ligands for affinity chromatography of aminopeptidases. At present a nonspecific (ion-exchange, hydrophobic) interaction of sorbent and aminopeptidases is observed, which necessitates to increase the specificity at the stage of the enzyme desorption in the further studies.