Specific Adsorbents for Affinity Chromatography of Benzodiazepine Binding Proteins

The use of affinity chromatography as means of isolating/ purifying proteins which have an affinity for benzodiazepine is described. Three such drugs are employed: chlorazepate, clonazepam and delorazepam. The results presented in this paper indicate that the proposed technique only works for chlorazepate and delorazepam. In fact these benzodiazepine–Sepharose derivatives are able to retain specifically proteins from human serum and rat kidney, lung, skeletal muscle and brain.     

Isolation of Putative Benzodiazepine Receptors from Rat Brain Membranes by Affinity Chromatography

Abstract: The benzodiazepine receptor from rat brain was solubilised and purified 5200-fold by affinity chromatography. The affinity column contained an immobilized benzodiazepine (delorazepam) and biospecific elution with 6 m m -chlorazepate was achieved. The purified receptor is apparently homogeneous in SDS-polyacrylamide gel electrophoresis. The native protein had a molecular weight of 240,000, and the subunit one of 60,000. The dissociation constant ( K _D) is 8 n m for [³H]diazepam. A correlation exists between the value of affinity obtained for benzodiazepine derivatives and their known pharmacological effectiveness.     

Human heat shock gene expression and the modulation of plasma membrane Na+, K+-ATPase activity

Benzodiazepine receptor solubilized from bovine cortical membranes was bound to a new benzodiazepine affinity column, the synthesis of which is described. Bio-specific elution with the benzodiazepine compound chlorazepate resulted in the elution of fractions highly enriched in specific binding for the GABA receptor agonist muscimol. Specific activity for [³H]muscimol binding was >1.3 nmol/mg protein. It is shown that [³H]flunitrazepam binding activity can be recovered by removal of chlorazepate from the purified fraction. These results strongly support a model which suggests that the 2 binding sites reside on the same physical entity.     

Microcalorimetric method to determine competitive binding. Action of a psychotropic drug (dipotassium chlorazepate) on L-tryptophan . human serum albumin complex.

A mathematical treatment and an original microcalorimetric method are developed to verify an eventual competitive binding between any two substances for the same macromolecule. To apply this method, a competitive binding of L-tryptophan and one benzodiazepin (dipotassium chlorazepate) for human serum albumin is perfectly demonstrated. The association constants and the enthalpy variations are equal to 14 000 +/- 2000 M-1 and --6.6 +/- 0.2 kcal/mol for human serum albumin . tryptophan complex and 13 000 +/- 1000 M-1 and --10.0 +/- 0.2 kcal/mol for human serum albumin . chlorazepate complex. In all cases the stoichiometry is equal to one. The binding of tryptophan to human serum albumin is partially stereospecific; the association constant and the enthalpy variation for D-tryptophan complex are equal, respectively, to 1000 +/- 200 M-1 and --2.6 +/- 0.3 kcal/mol.     

Microcalorimetric method to determine competitive binding action of a psychotropic drug (dipotassium chlorazepate) on L-tryptophan · human serum albumin complex

A mathematical treatment and an original microcalorimetric method are developed to verify an eventual competitive binding between any two substances for the same macromolecule. To apply this method, a competitive binding of L-tryptophan and one benzodiazepin (dipotassium chlorazepate) for human serum albumin is perfectly demonstrated.The association constants and the enthalpy variations are equal to 14 000 ± 2000 M^?1 and ?6.6 ± 0.2 kcal/mol for human serum albumin · tryptophan complex and 13 000 ± 1000 M^?1 and ?10.0 ± 0.2 kcal/mol for human serum albumin · chlorazepate complex. In all cases the stoichiometry is equal to one.The binding of tryptophan to human serum albumin is partially stereospecific; the association constant and the enthalpy variation for D-tryptophan complex are equal, respectively, to 1000 ± 200 M^?1 and ?2.6 ± 0.3 kcal/mol.     

Aminoalkyl affinity matrices.

Aminoalkyl matrices are used in affinity chromatography of amine oxidases and other proteins with affinity for amino groups. Under appropriate circumstances chromatography on aminoalkyl matrices may yield purification factors around 100 to 1000, and they have been used in affinity purification of many members of the amine oxidase family. Other proteins with affinity for aminoalkyl matrices include thiol ester proteins, lactoferrin, and proteins with lysine-binding kringles (plasminogen, plasminogen activator, apolipoprotein A). The affinity of thiol ester proteins for aminoalkyl matrices is abolished after inactivation of the thiol ester group by reaction with low molecular weight amines including ammonia. Due to this, an ammonium sulphate precipitation step should be included in purification schemes for amine oxidases. The affinity of lactoferrin for aminoalkyl matrices stems from an affinity for the repeating amino groups in glycosaminoglycans, and this explains why lactoferrin requires diamines for efficient elution. The affinity of plasminogen for aminoalkyl groups is exploited in a one-step purification from plasma, and is also utilised in purification schemes for angiostatin, an angiogenesis-inhibiting fragment of plasminogen. Apolipoprotein A is homologous to plasminogen, and also has affinity for aminohexyl columns. The common binding motif for these proteins are lysine-binding kringles. Due to the properties of the amino group itself, aminoalkyl matrices will inevitably also function as anion exchangers, and this must be taken into consideration in the choice of conditions for sample loading, column washing and elution of bound proteins. Depending on the length of the alkyl chain, the matrices also have a potential for hydrophobic interactions. This property has been exploited in the purification of several proteins but must be minimized during affinity chromatography of amine oxidases. In conclusion, aminoalkyl matrices are valuable tools for affinity chromatography of several different proteins, and simple variations of sample pretreatment, sample loading, and column washing and elution conditions allow efficient selective purification of proteins with different affinities for the matrices.     

Interaction of DnaK with native proteins and membrane proteins correlates with their accessible hydrophobicity

Molecular chaperones are involved in protein folding, protein targeting to membranes, and protein renaturation after stress. They interact specifically with hydrophobic sequences that are exposed in unfolded proteins, and buried in native proteins. We have studied the interaction of DnaK with native water-soluble proteins and membrane proteins. DnaK–native protein interactions are characterized by dissociation constants between 1 and 50 μM (compared with 0.01–1 μM for unfolded proteins). This affinity is within the range of most intracellular protein concentrations, suggesting that DnaK interacts with a greater number of native proteins than previously suspected. We found a correlation between the affinity of native proteins for DnaK and their affinity for hydrophobic-interaction chromatography adsorbents, suggesting that DnaK interacts with exposed hydrophobic groups in native proteins. The interaction between DnaK and membrane proteins is characterized by DnaK's high affinity for detergent-solubilized membrane proteins, and its lower affinity for membrane proteins inserted in lipid bilayers, suggesting that the chaperone can interact with the hydrophobic sequences of the former, while it cannot penetrate the hydrophobic core of lipid bilayers. Thus, the specificity of DnaK for hydrophobic sequences is involved in its interaction with not only unfolded proteins, but also native water-soluble proteins and membrane proteins. All proteins interact with DnaK according to their exposed hydrophobicity.     

Properties of flaky affinity resin with co-continuous structure

A new type of flaky affinity resin for capture of the target proteins was prepared to discuss its properties compared with those of a particulate affinity resin. The resin prepared had totally co-continuous structure (monolith) and was utilized in the shape of flake. The concentration of surface amino groups for immobilization of ligand was determined to be 22.3 micromol/ml. Immobilizations of ligand such as Sulfonamide, Ketoprofen, Captopril, or Methotrexate (MTX) on the affinity resin were quantitatively proceeded to afford fully covered (100%) affinity resins. Control of the immobilization rate of affinity resin using Sulfonamide or Ketoprofen was successfully achieved with the calculated immobilization rate. The flaked shape of affinity resin (100-400 microm) presumably simplified affinity experimental procedures and the affinity resin immobilizing Sulfonamide effectively captured one of the target proteins, CAII, without non-specifically bound proteins. The observed properties of the flaky affinity resin as well as ease in handling are really useful for capture of the target proteins of possible rare ligands.     

Morphogenetic messages are in the extracellular matrix: biotechnology from bench to bedside

The origin and evolution of multicellular metazoa was accompanied by the appearance of extracellular matrix. The demineralized extracellular matrix of bone is enriched in morphogenetic proteins that induce bone. Bone morphogenetic proteins (BMPs) are intimately bound to collagens. BMP-4 has high affinity for type-IV collagen, and other binding proteins such as noggin and chordin. Soluble morphogens are kept in the solid state by extracellular matrix. In this sense Nature used the principles of affinity matrices long before humans patented the principle of affinity chromatography.     

Isolation and characterization of Z-DNA binding proteins from wheat germ

The preparation of a heterogeneous non-histone protein extract from wheat germ utilizing Br-poly(dG-dC).poly(dG-dC) (Z-DNA) affinity chromatography is described. The binding characteristics of antibodies against Z-DNA are used as a model system to define important criteria that the DNA binding behavior of a Z-DNA binding protein should display. We show that the wheat germ extract contains DNA binding proteins specific for left-handed Z-DNA by these criteria. The affinity of the proteins measured by competition experiments was approximately 10(5) greater for Br-poly(dG-dC).poly(dG-dC) (Z-DNA) than for poly(dG-dC).poly(dG-dC) (B-DNA). The affinity of the proteins for plasmid DNA increases with increasing negative superhelicity which is known to stabilize Z-DNA. The proteins are shown to compete with Z-DNA antibodies for binding to supercoiled plasmids. Finally, the affinity for two plasmids at a given superhelical density is greater for the plasmid containing an insert known to form Z-DNA than for a plasmid without the insert. The proteins exhibit a 2-3-fold greater affinity for stretches of (dC-dA)n.(dT-dG)n over stretches of (dG-dC)n.(dG-dC)n when both sequences are induced to form Z-DNA by supercoiling.     

Affinity partitioning and extraction of proteins.

Affinity partitioning of enzymes and plasma proteins in aqueous two-phase systems has been reviewed. Besides basic theoretical considerations of the principle of affinity partitioning the chemistry of coupling ligands to the polymers, the nature and properties of selected biomimetic ligands like dye-ligands, immunoligands, metal chelate ligands and hydrophobic ligands are reported. The usefulness of affinity partitioning for studying the affinity of ligands and their specificity to proteins has been demonstrated by selected examples. The method proved also applicable to study the structural dynamics of proteins as exemplified with phosphofructokinase from baker's yeast and human alpha-2-macroglobulin. The current knowledge of metal chelate affinity partitioning is presented as well as the applicability of affinity partitioning for the purification of enzymes.     

Challenges and opportunities in the purification of recombinant tagged proteins

The purification of recombinant proteins by affinity chromatography is one of the most efficient strategies due to the high recovery yields and purity achieved. However, this is dependent on the availability of specific affinity adsorbents for each particular target protein. The diversity of proteins to be purified augments the complexity and number of specific affinity adsorbents needed, and therefore generic platforms for the purification of recombinant proteins are appealing strategies. This justifies why genetically encoded affinity tags became so popular for recombinant protein purification, as these systems only require specific ligands for the capture of the fusion protein through a pre-defined affinity tag tail. There is a wide range of available affinity pairs “tag-ligand” combining biological or structural affinity ligands with the respective binding tags. This review gives a general overview of the well-established “tag-ligand” systems available for fusion protein purification and also explores current unconventional strategies under development.     

Lectin binding characterstics of mouse epididymal fluid and sperm extracts

Glycoproteins from luminal fluid of the mouse cauda epiciidymidis have been compared with glycoproteins from Triton X-100 extracts of mouse spermatozoa from varying regions of the epididymis, using lectins with specific affinity for different sugar residues. Concanavalin A recognizes 11 glycocomponents on Western blots of fractionated caudal fluid; wheat germ agglutinin (WGA) binds 12 proteins; Ulex europaeus agglutinin (UEA) binds seven; and Dolichos biflorus agglutinin (DBA) recognizes nine. Several of these glycoproteins display an affinity for more than one lectin, indicating a diversity in their exposed carbohydrate residues; whereas other proteins bind only one of the four lectins used. The results also show that some glycoproteins exhibit a higher affinity for particular lectins. Eight glycoproteins of similar mobility and lectin-binding characteristics are detected in Triton X-100 extracts of spermatozoa from different regions of the epididymis and in caudal fluid. The lectin affinity of some proteins appears or increases in spermatozoa from distal epididymal regions (54 kD, 32 kD), whereas the lectin affinity of others decreases (29 kD, 40 kD). There are differences in lectin affinities between proteins in sperm extracts and in caudal fluid. Some proteins show an affinity for three or four lectins in caudal fluid, but proteins of similar electrophoretic mobility in sperm extracts bind only one or two of the lectins. These data show that glycoproteins of similar mobility are present in caudal fluid and in Triton-X-100 sperm extracts, implying a potential interaction between caudal fluid components and epididymal sperm.     

Deoxyribonucleic acid-binding proteins in vegetative Bacillus subtilis: alterations caused by stage O sporulation mutations.

Deoxyribonucleic acid (DNA)-binding proteins have been compared between wild-type Bacillus subtilis and five sporulation mutants blocked at different stage O loci. Extracts from exponentially growing cells have been fractionated for proteins binding to single-stranded calf thymus DNA-cellulose and double-stranded B. subtilis DNA-cellulose. In nutrient broth, stage O mutations cause an accumulation of proteins with affinity for double-stranded DNA. Suppression of the mutation with extragenic suppressors relieves the accumulation. In minimal glucose medium, the stage O mutations also cause accumulation of proteins with affinity for double-stranded DNA, but the species accumulated are different from those of nutrient broth-grown cells. In neither case did stage O mutations affect proteins with affinity for single-stranded DNA. The results suggest that the products of stage O loci are functional and operative during vegetative growth.     

Existence of different populations of the dendrotoxin I binding protein associated with neuronal K+ channels

The binding sites of dendrotoxin I, mast cell degranulating peptide, and beta-bungarotoxin are thought to be associated with neuronal K+ channels. The different binding sites seem to reside on the same molecular assembly as each toxin can allosterically inhibit the binding of the others. Affinity chromatography on a beta-BTX Aca 22 affinity column has shown that there is an heterogeneous population of dendrotoxin I binding proteins. Two subtypes were separated: DTXI binding proteins with low affinity for beta-BTX (60-70% of total) and DTXI binding proteins with high affinity for beta-BTX (30-40% of total). Binding of 125I-DTXI and 125I-MCD to the former subtype is inhibited by beta-BTX with a low affinity (IC50 = 560 nM), while inhibition at the latter subtype occurs with a high affinity (IC50 = 10-16 nM). The DTXI binding subtype with low affinity for beta-BTX contains most (85-90%) of the binding sites for 125I-MCD.     

Large-scale affinity purification — state of the art and future prospects

Enzymes, human plasma proteins and interferon are examples of proteins which are currently being isolated by large scale affinity purification methods. Although affinity chromatography is still the dominant affinity purification technique, others — such as batch affinity adsorption, affinity precipitation and affinity partitioning — offer certain advantages in large-scale work. Immunosorbents based on monoclonal antibodies and affinity adsorbents based on HPLC matrices are expected to be used more frequently on a production scale.     

A simple affinity spin tube filter method for removing high-abundant common proteins or enriching low-abundant biomarkers for serum proteomic analysis

Although it is possible to identify new proteins from crude cell extracts using proteomics technology, it is often difficult to elucidate low-abundant biomarkers in the presence of a large amount of high-abundant proteins in serum. We have developed a simple and rapid method using an affinity spin tube filter to remove high-abundant common proteins and enrich the low-abundant biomarkers. The affinity spin tube filter contains protein G, coupled with antibodies against either high-abundant proteins or specific proteins of interest. After incubating with serum, the flow-through or the elute was collected and analyzed by two-dimensional gel electrophoresis. By using this affinity spin tube filter, the possibilities of identifying new biomarkers are shown. This technique could be used for large-scale sample preparation for high-throughput proteomic analysis.     

Heparin–protein interactions: From affinity and kinetics to biological roles. Application to an interaction network regulating angiogenesis

Numerous extracellular proteins, growth factors, chemokines, cytokines, enzymes, lipoproteins, involved in a variety of biological processes, interact with heparin and/or heparan sulfate at the cell surface and in the extracellular matrix (ECM). The goal of this study is to investigate the relationship(s) between affinity and kinetics of heparin–protein interactions and the localization of the proteins, their intrinsic disorder and their biological roles. Most proteins bind to heparin with a higher affinity than their fragments and form more stable complexes with heparin than with heparan sulfate. Lipoproteins and matrisome-associated proteins (e.g. growth factors and cytokines) bind to heparin with very high affinity. Matrisome-associated proteins form transient complexes with heparin. However they bind to this glycosaminoglycan with a higher affinity than the proteins of the core matrisome, which contribute to ECM assembly and organization, and than the secreted proteins which are not associated with the ECM. The association rate of proteins with heparin is related to the intrinsic disorder of heparin-binding sites. Enzyme inhibitor activity, protein dimerization, skeletal system development and pathways in cancer are functionally associated with proteins displaying a high or very high affinity for heparin (K_D < 100 nM). Besides their use in investigating molecular recognition and functions, kinetics and affinity are essential to prioritize interactions in networks and to build network models as discussed for the interaction network established at the surface of endothelial cells by endostatin, a heparin-binding protein regulating angiogenesis.     

A versatile lentiviral expression system to identify mammalian protein-protein interactions

Protein-protein interactions (PPIs) are central to our understanding of protein function, biological processes and signaling pathways. Affinity purification coupled with mass spectrometry (AP-MS) is a powerful approach for detecting PPIs and protein complexes and relies on the purification of bait proteins using bait-specific binding reagents. These binding reagents may recognize bait proteins directly or affinity tags that are fused to bait proteins. A limitation of the latter approach is that expression of affinity tagged baits is largely constrained to engineered or unnatural cell lines, which results in the AP-MS identification of PPIs that may not accurately reflect those seen in nature. Therefore, generating cell lines stably expressing affinity tagged bait proteins in a broad range of cell types and cell lines is important for identifying PPIs that are dependent on different contexts. To facilitate the identification of PPIs across many mammalian cell types, we developed the mammalian affinity purification and lentiviral expression (MAPLE) system. MAPLE uses recombinant lentiviral technology to stably and efficiently express affinity tagged complementary DNA (cDNA) in mammalian cells, including cells that are difficult to transfect and non-dividing cells. The MAPLE vectors contain a versatile affinity (VA) tag for multi-step protein purification schemes and subcellular localization studies. In this methods article, we present a step-by-step overview of the MAPLE system workflow.