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An improved forward chemical genetics approach was successfully demonstrated using a tagged library concept. A small-molecule triazine library with linkers was used to screen for brain/eye developmental phenotypes in a zebrafish embryo system. This approach enabled the rapid isolation of the target proteins by facile affinity matrix preparation and elucidated the first small-molecule inhibitors for several ribosomal accessory proteins or their complex as the target.

Sirolimus (rapamycin), a new immunosuppressive drug, inhibits proliferation of a wide spectrum of T and B cells. The immunosuppressive mechanism of sirolimus is still unclear. We recently isolated a membrane associated protein with an apparent molecular weight of 210 kDa, p210, from cultured Molt 4 cells and BJAB cells and from normal human T cells using an affinity matrix method. The p210 binds to sirolimus:FKBP12 complex, but only at background levels to FKBP12 alone, to FK506:FKBP12 complex, or sirolimus-biotin alone. Among the sirolimus analogs tested, the binding ability of p210 to drug:FKBP12 complexes correlates with the immunosuppressive activity of the drugs, suggesting that p210 is the sirolimus effector protein.

New affinity purification techniques involving water-soluble biospecific polymers have been reported during the past 15 years, with the ambition of providing an alternative to affinity chromatography. This approach was prompted owing to certain limitations among which the difficulty to properly control the synthesis of the affinity matrix and the risk of non-specific binding of contaminants are directly related to the use of insoluble supports.

Publisher Summary This chapter discusses synthesis and applications of affinity matrix containing immobilized βγ subunits of G proteins. All G proteins composed of α,β, and γ subunits. Stimulation of the proteins is effected by exchange of GTP for GDP. This activation can be mimicked, in vitro, by the binding of AIF 4 - in concert with guanosine 5'-diphosphate (GDP). Activation promotes dissociation of α and βγ subunits. Because generic purified complexes of βγ subunits interact with a wide variety of unique α subunits, it is possible to use βγ as an affinity reagent for the study of α subunits. The development of a functional immobilized βγ resin provided a novel method for isolating and purifying α subunits of G proteins and a unique means for studying the interaction between α and βγ subunits. The immobilization of βγ subunits on ω-aminobutyl-agarose takes advantage of the relative insensitivity of the function of βγ subunits to modification by maleimides.

Two types of structural variants of d - myo -inositol 1,4,5-trisphosphate [Ins(1,4,5)P 3 ] were prepared by a chemo-enzymatic route. These 6- O -substituted analogues retained the biological activity of Ins(1,4,5)P 3 , and were able to elicit Ca 2+ release from porcine brain microsomes. Moreover, these derivatives allowed the preparation of Ins(1,4,5)P 3 -based immunogens and affinity matrix which were successfully applied to the preparation and purification of antibodies against Ins(1,4,5)P 3 . These antibodies displayed discriminative affinity towards Ins(1,4,5)P 3 , and provide a useful tool to study intracellular Ca 2+ mobilization.

New PtdIns(3,4,5)P3 binding proteins have been identified utilising PtdIns(3,4,5)P3 modified affinity matrix 1 which was synthesised from myo-inositol derivative 2, phosphoramidite 9 and an agarose based solid support.

Immobilized kinase inhibitors have emerged as powerful reagents for the determination of kinase inhibitor selectivity and for the enrichment of protein targets from cellular lysates. Here, we report the design and synthesis of a set of "clickable" 4-anilinoquinazoline kinase inhibitors. We demonstrate that the attachment of a flexible tether that contains a bio-orthogonal azide functionality does not adversely affect the potency or selectivity of these inhibitors. Furthermore, we demonstrate the utility of these inhibitors through the generation of an affinity matrix for the enrichment of interacting proteins from cellular lysates.

An NAD-matrix is capable of the separation of three contaminant reductase activities from highly purified beef heart cytochrome c oxidase. Non-specific binding of cytochrome c oxidase occurs as well. When the matrix is saturated with respect to the non-specific binding, continued binding of the reductases can be observed. As a consequence of equilibration with the matrix, the turnover numbers of the unbound cytochrome c oxidase are increased.

Phosphotransacetylase from Clostridium kluyveri was purified using a C8-(6-aminohexyl)-amino-desulfo-coenzyme A-Sepharose column. The method of synthesis of the affinity matrix is described. A crude extract was treated with ammonium sulfate and chromatographed on the desulfo-coenzyme A-Sepharose column. Using this method the enzyme was purified 83-fold and was found to be 73% pure. A new method for the determination of the purity of phosphotransacetylase by activity staining of polyacrylamide gels with 5,5′-dithiobis(2-nitrobenzoic acid) is described.

A procedure for separating Escherichia coli aminoacyl-tRNA from unacylated tRNA or components of the aminoacylation reaction, thereby achieving an aminoacyl-tRNA product with a very high specific activity, is described. The method utilizes the specific recognition of aminoacyl-tRNA for E. coli protein synthesis elongation factor Tu which has been immobilized on an affinity matrix. The application of the affinity procedure as a means of purifying a single aminoacyl-tRNA from an unfractionated mixture of tRNAs is also discussed.

The potential of affinity chromatography for the characterization of strong solute-ligand interactions is explored by studying the NADH-dependent elution of rabbit muscle lactate dehydrogenase from a column of trinitrophenyl-Sepharose in 0.067 M phosphate, pH 7.2. An interesting development is the simplification of the general affinity chromatography theory that emanates from the use of affinity matrices with a high concentration of immobilized reactant groups. The resultant expression allows evaluation of the intrinsic association constant for solute-ligand interactions from a single series of either zonal or frontal affinity chromatographic experiments conducted in the presence of a range of free ligand concentrations. Thus, contrary to previous belief, an affinity matrix designed for solute purification work should prove to be an asset for, rather than an impediment to, the study of solute-ligand interactions by quantitative affinity chromatography.

Glutathione reductase bound to an affinity matrix of immobilized GSSG was eluted by its coenzyme NADPH rather than by its substrate GSSG or by NADH. NADP+ could also elute the enzyme, but a high concentration was needed to release enzyme activity in a sharp peak. This chromatographic system exhibits an unusual form of biospecificity in which the enzyme is bound to an immobilized substrate but released by its soluble cofactor.

The possibility of separating pyruvylated polysaccharides into pyruvate-rich and pyruvate-poor fractions has been demonstrated using an affinity matrix. Thus in some polymers, a mixture of strand types exists. The affinity matrix was prepared by coupling antibodies to a Rhizobium polysaccharide to Sepharose gel. Elution was accomplished by the addition of pyruvate to the eluting buffer. Non-pyruvated polysaccharides were not adsorbed.

A simple and highly sensitive column chromatographic method based on specific chelation of o -dihydroxyphenols to dihydroxyboryl cellulose has been developed for quantitative isolation and concentration of catecholic substances from biological samples. At slightly alkaline pH, all catechols devoid of an acidic group adjacent to hydroxyl bind tightly to the boryl cellulose support. Upon derivatization of the carboxyl group, acidic catechols also bind to the column. Elution can be achieved with acidic buffer in the range of pH 2–4. Usefulness of this affinity matrix for the isolation of catechols from plant and insect materials and for concentration from dilute solutions is demonstrated.

A simple and rapid method for the purification of Peanut Agglutinin by affinity chromatography on cross-linked arabinogalactan is described. Cross-linked arabinogalactan shows a high capacity for PNA. The lectin has been obtained to electrophore-tic purity and has a high hemagglutinating specific activity.

Publisher Summary This chapter explains regulatory subunits of bovine heart and rabbit skeletal muscle cAMP-dependent protein kinase isozymes. Cyclic AMP-dependent protein kinase exists in two major isozymic forms, types I and II, which are present in varying relative concentrations, depending upon the tissue and species studied. Regulatory subunits from both isozymes can be isolated from a single tissue source when both types are present in approximately equal amounts, such as in porcine skeletal muscle. The choice of tissue and method of elution ultimately depends upon the quantity of R I or R II subunits needed and the nature of the studies for which they are to be used. The procedures described in the chapter for purification of R I and R II are based on relatively large amounts of starting material. If scaling down is appropriate, the final volume of cAMP-binding material which is eluted from diethylaminoethyl (DEAE)-cellulose may be small enough that concentration by (NH 4 ) 2 SO 4 before applying it to the affinity matrix is unnecessary. It is advisable to centrifuge all material to prevent blockage of the column. Omitting the (NH 4 ) 2 SO 4 precipitation step saves at least 6 hours and makes it possible to go from intact tissue to the cAMP-affinity column in

Purification of glutathione S -transferases A and C from rat liver cytosol using an affinity matrix coupled with cholic acid is described. The method provides a convenient means for the rapid and homogeneous preparation of both transferases.

Limited structural information has hitherto been obtainable from crosslinking studies on purified proteins. A major limitation has been the lack of a procedure permitting confident interpretation of “negative” crosslinking data. The “spreading-out” of a protein on an affinity matrix at a critically low density below which intermolecular bridge formation does not occur, prior to reaction with a chemical crosslinker (which may be safely added in high concentrations), provides an approach which yielded valuable information regarding the tertiary and quaternary structure of concanavalin A. This information could be cross-checked from the existing data on this protein previously obtained by crystallography and other biophysical techniques, thus demonstrating the validity of this probe. This matrix approach promises to be particularly useful in the structural study of poorly soluble membrane-bound proteins and other proteins which are difficult to crystallise.

The 90 000-Mr glucocorticoid receptor was purified to homogeneity according to sodium dodecylsulfate/polyacrylamide gel electrophoresis. An affinity column containing either dexamethasone-17 beta-carboxylic acid or dexamethasone-21-methanesulfonate bound to the matrix with the help of a disulfide bond is used in this study. Using this affinity matrix, in a single step, 8700-fold purification of glucocorticoid receptor from rat liver cytosol could be achieved. Following the method of activation and DNA-cellulose chromatography the 90 000-Mr receptor subunit was purified to homogeneity.

Epoxy Sepharose, an activated affinity matrix which has been used for immobilisation of carbohydrates has been tried for immobilisation of proteins. Under normal conditions of coupling at neutral or alkaline pH proteins do not couple to epoxy Sepharose. However, a very high salt concentration during coupling allows the binding of proteins to epoxy Sepharose at a pH as low as 8·5. Increasing ionic strength and/or pH facilitates the binding. The bioactivity of the proteins is not destroyed by the immobilisation. This matrix, unlike cyanogen bromide-Sepharose, retains its ability to bind albumin by 80–90%even after 60 days of storage in aqueous suspension at $4^0C$. Its capacity to bind proteins is comparable to that of cyanogen bromide-Sepharose.

Insoluble polystyrenes substituted with sulphonate and l -arginyl methyl ester have been synthesized. Using their specific affinity for thrombin, we developed a simple one-step chromatographic procedure for thrombin purification. As a control, insoluble polystyrenes substituted only with sulphonate groups were tested. The results obtained confirmed the importance of the arginyl residues grafted onto these polymers to obtain an affinity matrix useful for purifying thrombin with a high specific activity and a good recovery.

Some epigenetic modifier proteins have become validated clinical targets. With a few small molecule inhibitors already approved by national health administrations and many more in the pharmaceutical industry pipelines, there is a need for technologies that can promote full comprehension of the molecular action of these drugs. Proteomics, with its relatively unbiased nature, can contribute to a thorough understanding of the complexity of the megadalton complexes, which write, read and erase the histone code, and it can help study the on-target and off-target effect of the drugs designed to modulate their action. This review on the one hand gathers the published affinity probes able to decipher small molecule targets and off-targets in a close-to-native environment. These are small molecule analogues of epigenetic drugs conceived as protein target enrichment tools after they have engaged them in cells or lysates. Such probes, which have been designed for deacetylases, bromodomains, demethylases, and methyltransferases not only enrich their direct protein targets but also their stable interactors, which can be identified by mass spectrometry. Hence, they constitute a tool to study the epigenetic complexes together with other techniques also reviewed here: immunoaffinity purification with antibodies against native protein complex constituents or epitope tags, affinity matrices designed to bind recombinantly tagged protein, and enrichment of the complexes using histone tail peptides as baits. We expect that this toolbox will be adopted by more and more researchers willing to harness the spectacular advances in mass spectrometry to the epigenetic field.

Arabinogalactan has been cross-linked to give a new high capacity affinity matrix for the purification of Ricinus communis lectins. It shows a capacity which is 50 times greater than that of the conventional affinity matrices.