Affinity chromatography: specific binding of hemoglobin on agarose linked haptoglobin

Human haptoglobin was coupled to agarose and used as affinity adsorbant to bind human hemoglobin. The optimal conditions of hemoglobin binding and dissociation were defined. It was found that the haptoglobin adsorbant removed effectively and specifically free hemoglobin from hemolysed sera.     

Manufacturing of peptides exhibiting biological activity

Numerous studies have shown that food proteins may be a source of bioactive peptides. Those peptides are encrypted in the protein sequence. They stay inactive within the parental protein until release by proteolytic enzymes (Mine and Kovacs-Nolan in Worlds Poult Sci J 62(1):87–95, 2006; Hartman and Miesel in Curr Opin Biotechnol 18:163–169, 2007). Once released the bioactive peptides exhibit several biofunctionalities and may serve therapeutic roles in body systems. Opioid peptides, peptides lowering high blood pressure, inhibiting platelet aggregation as well as being carriers of metal ions and peptides with immunostimulatory, antimicrobial and antioxidant activities have been described (Hartman and Miesel in Curr Opin Biotechnol 18:163–169, 2007). The biofunctional abilities of the peptides have therefore aroused a lot of scientific, technological and consumer interest with respect to the role of dietary proteins in controlling and influencing health (Möller et al. in Eur J Nutr 47(4):171–182, 2008). Biopeptides may find wide application in food production, the cosmetics industry as well as in the prevention and treatment of various medical conditions. They are manufactured by chemical and biotechnological methods (Marx in Chem Eng News 83(11):17–24. 2005; Hancock and Sahl in Nat Biotechnol 24(12):1551–1557, 2006). Depending on specific needs (food or pharmaceutical industry) different degrees of peptide purifications are required. This paper discusses the practicability of manufacturing bioactive peptides, especially from food proteins.     

Affinity chromatography using enzymatically synthesized nucleotide-containing DNA binding polymers

A series of nucleotide-containing polyphenols has been synthesized by a simple, two-step enzymatic method. The binding properties of these synthetic polymers to complementary oligonucleotides have been evaluated using a commercially available oligo(dT)cellulose column. Complementary synthetic nucleosides were retained on this column to a greater extent than non-complementary synthetic nucleosides. These results suggest that the synthetic nucleosides prepared via this two-step enzymatic approach may have application as affinity matrices.     

Biotechnology techniques for the development of new tumor specific peptides

Peptides, proteins and antibodies are promising candidates as carriers for radionuclides in endoradiotherapy. This novel class of pharmaceuticals offers a great potential for the targeted therapy of cancer. The fact that some receptors are overexpressed in several tumor types and can be targeted by small peptides, proteins or antibodies conjugated to radionuclides has been used in the past for the development of peptide endoradiotherapeutic agents such as ⁹⁰Y-DOTATOC or radioimmunotherapy of lymphomas with Zevalin. These procedures have been shown to be powerful options for the treatment of cancer patients.Design of new peptide libraries and scaffolds combined with biopanning techniques like phage and ribosome display may lead to the discovery of new specific ligands for target structures overexpressed in malignant tumors. Display methods are high throughput systems which select for high affinity binders. These methods allow the screening of a vast amount of potential binding motifs which may be exposed to either cells overexpressing the target structures or in a cell-free system to the protein itself. Labelling these binders with radionuclides creates new potential tracers for application in diagnosis and endoradiotherapy. This review highlights the advantages and problems of phage and ribosome display for the identification and evaluation of new tumor specific peptides.     

Affinity adsorbent based on combinatorial phage display peptides that bind alpha-cobratoxin

Combinatorial phage display was used to discover peptides that selectively bind to the alpha-cobratoxin (neurotoxin) component of the multi-component venom of the Thai cobra, Naja kaouthia. Peptide sequences determined in this way were synthesized chemically and were covalently attached to agarose through the alpha-amino terminus. Such affinity chromatography supports selectively bound the alpha-cobratoxin component from crude venom, while passage of the crude venom over the support selectively depleted the venom of this component. The selective binding of alpha-cobratoxin to peptide-based solid-phase supports suggests that a limitless variety of peptides similarly obtained by combinatorial phage display can be used to craft specific analytical and preparative tools.     

Effect of preparations exhibiting cytokinin-like activity on the specific density of leaf in grasses

The effects of synthetic preparations exhibiting cytokinin-like activity (6-benzylaminopurine, Thidiazuron, and kartolin-2) on the specific leaf area (SLA) were studied in plants of the family Gramineae (wheat, Triticum aestivum L.; meadow fescue, Festuca pratensis Huds.; and reed fescue, F. arindinacea Schreb.). At the early stages of ontogeny (until the leaf area reached 50-60% of the maximum value), treatment of plants of the three species with cytokinin-like preparations caused an increase in SLA. The SLA value in these plants was correlated with the rate of photosynthetic assimilation of carbon dioxide and activities of carbon metabolism enzymes: ribulose-1,5-bisphosphate carboxylase/oxygenase (EC 4.1.1.39), NAD-malate dehydrogenase (EC 1.1.1.37), and NADP-glyceraldehydrophosphate dehydrogenase complex, which includes phosphoglycerate kinase (EC 2.7.2.3) and glyceraldehydrophosphate dehydrogenase (EC 1.2.1.13). However, there was no correlation of SLA with the activity of phospho(enol)pyruvate carboxylase (EC 4.1.1.31), an anaplerotic carboxylation enzyme of grasses. SLA is suggested to reflect the state and activity of the photosynthetic apparatus and can be recommended as a characteristic of photosynthesis variability (e.g., caused by cytokinin-like preparations).     

Affinity chromatography of aminoacyl-tRNA synthetases on specific tRNA columns without prior purification of tRNA

A method is described for the purification of aminoacyl-tRNA synthetases by affinity chromatography, using a column of tRNA lacking the cognate tRNA, followed by a column of the cognate tRNA. The ability of the enzyme to discriminate between cognate and non-cognate tRNA is exploited in a novel and rapid preparation of the two columns.     

Flavin binding by bacterial luciferase: Affinity chromatography of bacterial luciferase

Immobilized FMN covalently attached to Sepharose-6B-hexanoate binds bacterial luciferase. Immobilized flavin is also effective in its reduced form as a substrate in the light emitting reaction catalyzed by luciferase.     

Affinity chromatography of a sequence-specific DNA binding protein using Teflon-linked oligonucleotides

An affinity matrix was constructed by synthesis of a DNA oligonucleotide on a Teflon fiber support followed by deblocking and hybridization of the complementary strand. It was used to purify a sequence-specific binding protein at least 100-fold to near homogeneity. This matrix is easily fabricated on an automated DNA synthesizer, contains high levels of attached DNA, and has superior mechanical properties. It should be generally useful for affinity chromatography of DNA binding proteins.     

Affinity binding of inclusion bodies on supermacroporous monolithic cryogels using labeling with specific antibodies

A new chromatographic method based on affinity supermacroporous monolithic cryogels is developed for binding and analyzing inclusion bodies during fermentation. The work demonstrated that it is possible to bind specific IgG and IgY antibodies to the 15 and 17 amino acids at the terminus ends of a 33 kDa target protein aggregated as inclusion bodies. The antibody treated inclusion bodies from lysed fermentation broth can be specifically retained in protein A and pseudo-biospecific ligand sulfamethazine modified supermacroporous cryogels. The degree of binding of IgG and IgY treated inclusion bodies to the Protein A and sulfamethazine gels are investigated, as well as the influence of pH on the sulfamethazine ligand. Optimum binding of 78 and 72% was observed on both protein A and sulfamethazine modified cryogel columns, respectively, using IgG labeling of the inclusion bodies. The antibody treated inclusion bodies pass through unretained in the sulfamethazine supermacroporous gel at pH that does not favour the binding between the ligand on the gel and the antibodies on the surface of inclusion bodies. Also the unlabeled inclusion bodies went through the gel unretained, showing no non-specific binding or trapping within the gel. These findings may very well be the foundation for the building of a powerful analytical tool during fermentation of inclusion bodies as well as a convenient way to purify them from fermentation broth. These results also support our earlier findings [Kumar, A., Plieva, F.M., Galaev, I.Yu., Mattiasson, B., 2003. Affinity fractionation of lymphocytes using a monolithic cyogel. J. Immunol. Methods 283, 185-194] with mammalian cells that were surface labeled with specific antibodies and recognized on protein A supermacroporous gels. A general binding and separation system can be established on antibody binding cryogel affinity matrices.     

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.     

Affinity chromatography of arabinogalactan-proteins

Arabino-(1→3), (1→6)-β-d-galactan-proteins (AGPs) and related compounds from Lolium multiflorum (ryegrass) endosperm cell suspension culture, wheat endosperm, larchwood and Gladiolus stigma extract were shown to bind selectively at neutral pH to a column of Sepharose to which the anti-galactan myeloma protein J539 had been covalently linked, Elution was achieved with buffer at pH 3 or with a pulse of p-nitrophenyl-β-d-galactopyranoside at neutral pH. These observations formed the basis for an affinity chromatographic purification of AGPs from natural sources. Some heterogeneity in a ryegrass AGP preparation was indicated by its incomplete elution by d-galactose.     

Affinity chromatography of neoglycoproteins

Glycoproteins, as a class of biomolecules, exhibit much more heterogeneous structures than non-glycosylated proteins. They present a challenging area of research. Model glycoproteins with well-defined protein and carbohydrate structures are helpful in the search for high-resolution methods for the separation of glycoproteins. Neoglycoproteins, maltose-modified chymotrypsin and lactose-modified chymotrypsin, were synthesised by modifying chymotrypsin with maltose and lactose, respectively, using the reductive amination method. Boronate chromatography was applied to isolate the neoglycoproteins from non-glycosylated substances. The use of Tris–HCl as a shielding reagent during the boronate chromatography proved to be efficient in eliminating unwanted interactions between the boronate ligand and the peptide backbone of chymotrypsin. The retention time of neoglycoproteins on the boronate column was increased with increasing the degree of modification.     

Affinity chromatography of nucleases

The review concerns isolation and purification of nucleases by affinity chromatography. Different stationary ligands and the methods for their immobilization on supports are described, along with diverse eluents and various procedures for a nuclease detachment from the affinity sorbents. The data on the affinity chromatography application for measuring the dissociation constants of the enzyme complexes with either immobilized or soluble ligands are compiled.     

Affinity chromatography of human gamma-thrombin on fibrin-agarose

gamma-Thrombin was produced during autolysis or limited proteolysis of coagulant gamma-thrombin. This thrombin form loses its ability to coagulate fibrinogen but preserves the esterase and amidase activity on the low-molecular-weight synthetic substrates. This evidences for the integrity of the active site of gamma-thrombin and for the integrity break of the enzyme molecule region responsible for the binding with fibrinogen. gamma-Thrombin with the minimal coagulating activity, possessing high esterase and amidase activity is obtained. Fibrin-agarose possessing affinity to gamma-thrombin and specifically not binding gamma-thrombin was used to remove admixtures of the coagulant gamma-thrombin from the preparations of gamma-thrombin obtained during the enzyme autolysis.