Selective isolation and purification of tat protein via affinity membrane separation

This work deals with the separation of Tat protein from a complex fermentation broth using an affinity membrane system. Tat is a regulatory protein that is critical for HIV-1 replication and thus a potential candidate for vaccine and drug development. Furthermore, Tat can facilitate transport of exogenous molecules across cell membranes and is implicated in pathogenesis of HIV dementia. Affinity membranes were prepared through coupling of avidin within a 4-stack membrane construct. Tat (naturally biotinylated) accessibility in the bacterial lysate feed was influenced by the presence of RNAse, protein concentration, and ionic strength. Enhanced accessibility translated to a marked increase in the overall product yield per pass. The purity of the membrane-isolated Tat was compared to that prepared via packed column chromatography through SDS-PAGE, Western blot, activity assay, and neurotoxicity studies. Tat protein produced via membrane separation yielded primarily monomeric forms of the oligopeptide sequence, whereas column chromatography produced predominately polymeric forms of Tat. These differences resulted in changes in the neurotoxicity and cellular uptake of the two preparations.     

Tagged polymerase chain reaction subtractive hybridization for the enrichment of phage display random peptide libraries

Affinity selection of phage display peptide libraries is routinely used for isolating peptides capable of binding a range of molecules, including antibodies and receptors. This process is most successful when the selecting molecule is relatively pure, for example, a monoclonal antibody. However, isolation of peptides able to bind to target molecules present in a complex mixture is more difficult because the affinity selection process isolates peptides capable of binding to all molecules present in the mixture. Here we describe the development of a tagged polymerase chain reaction (PCR) subtractive hybridization method that is universally applicable for the targeted isolation of peptides able to bind to unique molecules within a complex mixture. We also describe a discriminatory limiting dilution PCR method that can be used to optimize hybridization conditions.     

Potential molecular chaperones involved in laminin chain assembly

To explore potential molecular chaperones involved in the intracellular assembly of laminin chains, bovine aortic endothelial cells were treated with a thiol cleavable divalent cross-linking reagent, dithio-bis-(succinimidylpropionate), and cellular proteins cross-linked to laminin chains were co-immunoprecipitated with anti-laminin antiserum. Sodium dodecylsulfate (SDS) gel electrophoresis of the precipitate under reducing condition showed polypeptides with estimated sizes of 80, 60 and 50 kDa together with laminin chains. Two dimensional electrophoresis, in which non-reducing and reducing SDS electrophoresis were combined, suggested that many molecules of these polypeptides were cross-linked to each laminin chain. Sepharose CL-4B beads conjugated with E8 fragment of mouse laminin-1 was prepared. Affinity chromatography with the beads of microsomal proteins from rat liver showed that Bip and HSP70 associated to laminin chains and dissociated upon ATP hydrolysis. Protein-disulfide isomerase also showed affinity to the column. GRP94 and calnexin showed strong affinity and were washed out only with a detergent solution. Thus, many molecular chaperones are suggested to be involved in the intracellular assembly of laminin chains. This revised version was published online in June 2006 with corrections to the Cover Date.     

Applications of affinity chromatography to the study of drug-melanin binding interactions

This short review reports on progresses in the study of drug-melanin interactions using the technique of affinity chromatography. Melanins are natural or synthetic pigments derived from the oxidation and polymerization of various precursors including L-dopa, tyrosine and cystein. Accumulation of toxic compounds, drugs, and metal ions in pigmented tissues through reversible binding to melanin has been linked to chronic toxicity. Affinity chromatography using chromatographic stationary phases based on physically adsorbed or chemically bonded melanin provides a useful tool for studying the interactions of small molecules and metal ions with melanin     

A method for the covalent capture and screening of diverse small molecules in a microarray format

This protocol describes a robust method for the covalent capture of small molecules with diverse reactive functional groups in microarray format, and outlines a procedure for probing small-molecule microarrays (SMMs) with proteins of interest. A vapor-catalyzed, isocyanate-mediated surface immobilization scheme is used to attach bioactive small molecules, natural products and small molecules derived from diversity-oriented synthesis pathways. Additionally, an optimized methodology for screening SMMs with purified proteins and cellular lysates is described. Finally, a suggested model for data analysis that is compatible with commercially available software is provided. These procedures enable a platform capability for discovering novel interactions with potential applications to immunoglobulin profiling, comparative analysis of cellular states and ligand discovery. With the appropriate materials and experimental setup, the printing of SMMs can be completed in 14 hours over 3 days. Screening and data analysis requires 2 days. A detailed timeline is provided.     

Affinity precipitation an option for early capture in bioprocessing

Affinity precipitation is a bioseparation technique where the affinity ligand is coupled to a stimuliresponsive polymer. Stimuli-responsive polymers show abrupt, yet reversible, phase transition (precipitation) in response to a small change in an environmental parameter. The corresponding ligand conjugates can be used to co-precipitate and thereby capture and isolate target molecules from complex solutions such as culture supernatants and cell lysates. The approach is compatible with a 'discardibles only' type of downstream process and can be scaled over several orders of magnitude. This report discusses the set-up and development of affinity precipitation procedures, the related instrumentation and scale up, as well as applications for the isolation of proteins and polynucleotides.     

Screening for small molecule inhibitors of embryonic pathways: sometimes you gotta crack a few eggs

Extract prepared from Xenopus eggs represents a cell-free system that has been shown to recapitulate a multitude of cellular processes, including cell cycle regulation, DNA replication/repair, and cytoskeletal dynamics. In addition, this system has been used to successfully reconstitute the Wnt pathway. Xenopus egg extract, which can be biochemically manipulated, offers an ideal medium in which small molecule screening can be performed in near native milieu. Thus, the use of Xenopus egg extract for small molecule screening represents an ideal bridge between targeted and phenotypic screening approaches. This review focuses on the use of this system for small molecules modulators of major signal transduction pathways (Notch, Hedgehog, and Wnt) that are critical for the development of the early Xenopus embryo. We describe the properties of Xenopus egg extract and our own high throughput screen for small molecules that modulate the Wnt pathway using this cell-free system. We propose that Xenopus egg extract could similarly be adapted for screening for modulators of the Notch and Hedgehog pathways.     

Superselective labelling of proteins: approaches and techniques

Affinity labelling is a popular method used for the study of macromolecules and their interactions with ligands. The method is based on the targeted delivery of a chemically cross-linkable group, attached to a reactive molecule with affinity for a particular site in the biopolymer of interest. In complex multicomponent systems, the applications of affinity labelling are restricted by the tendency of the reagents to randomly label nontargetted molecules. This review highlights techniques developed to minimize non-specific cross-linking and to achieve high selectivity for the labelling of target protein. Such techniques might be termed 'superselective labelling', as opposed to traditional, less selective approaches.     

Novel protein a affinity matrix prepared from two-dimensional protein crystals

In this article, we describe a novel type of affinity matrix which was prepared by covalently binding Protein A to crystalline cell surface layers (S-layers) from the gram-positive Clostridium thermohydrosulfuricum L111-69. S-layers were used in the form of cell wall fragments, which were obtained by breaking whole cells by ultrasonification and removing the cell content and the plasma membrane. In these thimble shaped structures, revealing a size of 1 to 2 mum, the peptidoglycan-containing layer was covered on both faces with a hexagonally ordered S-layer lattice composed of identical glycoprotein subunits. After crosslinking the S-layer protein with glutaraldehyde, carboxyl groups from acidic amino acids were activated with carbodiimide and used for immobilization of Protein A. Quantitative determination confirmed that up to two Protein A molecules were bound per S-layer subunit leading to a dense monomolecular coverage of the immobilization matrix with the ligand.Affinity microparticles were capable of adsorbing lgG from solutions of purified preparations, from artificial lgG-albumin mixtures, and from serum. The amount of lgG bound to affinity microparticles corresponded to the theoretical saturation capacity. Under appropriate conditions, up to 95% of the adsorbed lgG could be eluted again. Affinity microparticles were found to have an extremely low Protein A leakage and a high stability toward mechanical forces. Because pores in the S-layer lattice revealed a size of 4 to 5 nm, immobilization of Protein A and adsorption of lgG was restricted to the outermost surface area. This excludes mass transfer problems usually encountered with affinity matrices prepared from amorphous polymers where more than 90% of the ligands are immobilized in the interior. (c) 1994 John Wiley & Sons, Inc.     

Affinity chromatography purification of penicillinase of Bacillus licheniformis 749-C and its use to measure tuurnover of the cell bound enzyme

Affinity chromatography purification of small amounts of penicillinase using cephalosporin C covalently linked to Sepharose 4B has been used in examining the turnover of cell-bound penicillinase by B. licheniformis 749/C. Under conditions in which most of the nascent penicillinase is retained by the cells, turnover could be demonstrated (15% of the cell-bound enzyme in 2.5 hours), although most of the secreted enzyme is derived from newly-formed molecules.     

Affinity cross-flow filtration: purification of IgG with a novel protein a affinity matrix prepared from two-dimensional protein crystals

In this article, we describe the use of 1- to 2-mum sized affinity microparticles for the isolation and purification of IgG from artificial IgG-human serum albumin mixtures and clarified hybridoma cell culture supernatants by affinity cross-flow filtration. Affinity microparticles were prepared from cell wall fragments of Clostridium thermohydrosulfuricum L111-69, in which the peptidoglycan-containing layer was completely covered with a hexagonally ordered S-layer lattice. After crosslinking the S-layer protein with glutaraldehyde, carboxyl groups from acidic amino acids were activated with carbodiimide and used for immobilization of Protein. A. Quantitative determination confirmed that Protein A molecules formed a monomolecular layer on the outermost surface of the S-layer lattice. Affinity microparticles were found to withstand high centrifugal and shear forces and revealed no Protein A leakage or S-layer protein release under cross-flow conditions between pH 2 to 12. The IgG-binding capacity of affinity microparticles was investigated under crossflow conditions and compared with that obtained in batch adsorption processes. (c) 1994 John Wiley & Sons, Inc.     

Chemical tagging of a drug target using 5-sulfonyl tetrazole

Irreversible modification is one of the most promising strategies to identify cellular receptors of bioactive small molecules. Here we report that receptor proteins can be chemically tagged using a 5-sulfonyl tetrazole probe. 5-Sulfonyl tetrazole easily accepted nucleophilic attack of thiol groups, while 5-sulfinyl tetrazole did not. These functional groups were introduced into probe molecules of a natural product. Cyclosporine A, an immunosuppressant produced by a microbe, was derivatized to possess 5-sulfonyl tetrazole and a tag group, which enabled chemical tagging of cyclophilin A, the cellular receptor of cyclosporine A. Cyclosporine A derivative possessing 5-sulfinyl tetrazole could not tag cyclophilin A. This technique will allow efficient identification of cellular receptors of bioactive small molecules.     

A new efficient method of generating photoaffinity beads for drug target identification

Affinity purification is one of the most prevalent methods for the target identification of small molecules. Preparation of an appropriate chemical for immobilization, however, is a tedious and time-consuming process. A decade ago, a photoreaction method for generating affinity beads was reported, where compounds are mixed with agarose beads carrying a photoreactive group (aryldiazirine) and then irradiated with ultraviolet light under dry conditions to form covalent attachment. Although the method has proven useful for identifying drug targets, the beads suffer from inefficient ligand incorporation and tend to shrink and aggregate, which can cause nonspecific binding and low reproducibility. We therefore decided to craft affinity beads free from these shortcomings without compromising the ease of preparation. We herein report a modified method; first, a compound of interest is mixed with a crosslinker having an activated ester and a photoreactive moiety on each end. This mixture is then dried in a glass tube and irradiated with ultraviolet light. Finally, the conjugates are dissolved and reacted with agarose beads with a primary amine. This protocol enabled us to immobilize compounds more efficiently (approximately 500-fold per bead compared to the original method) and generated beads without physical deterioration. We herein demonstrated that the new FK506-immobilized beads specifically isolated more FKBP12 than the original beads, thereby proving our method to be applicable to target identification experiments.     

Improving success rates for lead generation using affinity binding technologies

Affinity technologies have been applied at several stages of the drug discovery process, ranging from target identification and purification to the identification of preclinical candidates. The detection of ligand-macromolecule interactions in lead discovery is the best studied and most powerful of these techniques. Although affinity methods have been in widespread use for about a decade, only recently have many reports emerged on their utility. Primary affinity screens of large libraries of small molecules or fragments have begun to produce results for challenging targets. Furthermore, in secondary assays affinity methods are opening new avenues to tackle important medicinal chemistry tasks.     

Microarray-based target identification using drug hypersensitive fission yeast expressing ORFeome

Identification of the cellular target of small molecules is a major challenge to developing biological tools and drug leads. Here we report a novel microarray-based system for identification of the target or the target pathway of small molecules using a set of drug-hypersensitive fission yeast strains that collectively overexpress each gene in the open reading frame-ome. The major advantage of this method is that it provides genome-wide interrogation but requires a relatively small amount of the test compound. Using this system, we identified 28 genes linked to etoposide sensitivity, which included genes for the drug target topoisomerase II and other plausible factors that regulate etoposide tolerance. Thus, our approach can accelerate the process of target identification of small molecules, which has the potential to reveal highly conserved genes of clinical relevance.     

Complement fixation activity of immunoglobulin G in ethylene glycol solutions

Affinity of IgG to the first complement factor C1q was found out to increase in 10-30% glycol solutions. Analytical ultracentrifugation and turbidity data showed that IgG molecules do not aggregate at such concentrations of glycol. The complement-binding effect may be caused by a conformational transition in the IgG molecules.     

Chitin Coated Cellulose as an Adsorbent of lysozyme like Enzymes Preparation and Properties

As a new adsorbent of lysozyme-like enzymes, chitin coated (CC-)cellulose was prepared. CC-cellulose was stable and had good flow properties for use in column chromatography. Affinity chromatography with CC-cellulose showed that 3~5 mg of lysozyme/ml resin was adsorbed specifically and desorbed quantitatively under mild conditions. The utilities of the method of affinity chromatography with CC-cellulose are discussed.     

Purification of anti-bromelain antibodies by affinity precipitation using pNIPAm-linked bromelain

Affinity precipitation has emerged as a very useful technique for the purification of proteins. Here it has been employed for the purification of anti-bromelain antibodies from rabbit serum. A system has been developed for reversibly binding and thermoprecipitating antibodies. Anti-bromelain antibodies were raised in rabbit by immunizing it with bromelain. Poly-N-isopropylacrylamide (pNIPAm)–bromelain conjugate was prepared and incubated with rabbit serum. After that the temperature was raised for thermal precipitation of the polymer. Antibodies were then eluted from the complex by incubating it with a small volume of buffer, pH 3.0. This method is very effective in concentrating the antibodies. Purity and specificity of the antibodies were checked by gel electrophoresis and enzyme-linked immunosorbent assay (ELISA), respectively. The study of the effect of pH and temperature on the binding of the antibodies to the conjugate showed that the optimum binding occurred at pH 8.0 and 25°C.The polymer enzyme conjugate was further used for another cycle.