Synthese von {2-[3-(2-Ammonioethoxy)benzoyl]ethyl}-trimethylammonium-Bromid-hydrobromid für die Affinitätschromatographie von Cholin-Acetyltransferase (ChAT; EC 2.3.1.6)

Synthesis of {2-[3-(2-Amonioethoxy)benzoyl]ethyl}trimethylammonium Bromide Hydrobromide for the Affinity Chromatography of Choline Acetyltransferase (ChAT; EC 2.3.1.6) ChAT is an essential enzyme that catalyzes the synthesis of acetylcholine from choline and activated acetic acid. For the affinity chromatography or biospecific adsorption, only those ligands are suited, which resemble the natural substrate. The ligands, however, cannot be bound directly to the matrix, but must be separated by a covalently bound spacer. Contrary to the often cited desorption by ionic gradients, we used the most specific desorption by the same ligand. The synthesis comprises seven steps starting from 3-hydroxyacetophenon ( 7 ).     

Application of the FCP-activation procedure to the synthesis of a biospecific adsorbent for trypsin

The synthesis of a biospecific adsorbent for trypsin was chosen as a model to investigate the applicability of FCP activation in affinity chromatography.p-Aminobenzamidine was chosen as a ligand, directly suitable for immobilization. The nonspecific binding properties of the first series of synthesized agarose derivatives were obviated either by FCP activation of the ligand instead of the matrix, or by modifying the initial FCP-activation procedure. The adsorbents prepared in this way, however, demonstrated no selectivity between trypsin and chymotrypsin. The introduction ofe-aminocaproic acid as a spacer was ineffectual. These problems were solved by the application of glycylglycine as a spacer. The final affinity matrices had a degree of substitution of approximately 4 μ.mol of ligand per gram gel (100 μmol ligand per gram dry adsorbent). The specific activity of a current trypsin preparation was increased by 58% in a single cycle. The biospecificity of these adsorbents was demonstrated.     

The quest for affinity chromatography ligands: are the molecular libraries the right source?

Affinity chromatography separations of proteins call for highly specific ligands. Antibodies are the most obvious approach; however, except for specific situations, technical and economic reasons are arguments against this choice especially for preparative purposes. With this in mind, the rationale is to select the most appropriate ligands from collections of pre‐established molecules. To reach the objective of having a large structural coverage, combinatorial libraries have been proposed. These are classified according to their nature and origin. This review presents and discusses the most common affinity ligand libraries along with the most appropriate screening methods for the identification of the right affinity chromatography selective structure according to the type of library; a side‐by‐side comparison is also presented.     

亲和色谱中配基的筛选与应用

亲和配基的选择与筛选是发展新的亲和色谱填料或构建一个新的亲和色谱体系所必须解决的首要问题。该文结合作者所在实验室的工作,对配基的选择、筛选与应用方面的一些进展进行了简要评述。作者所在实验室针对特定蛋白质和多肽的多肽亲和配基的筛选,开展了反义肽简并性的研究,发展了基于反义肽的组合化学筛选新方法。与常规的组合合成法相比,该方法简单、快捷、有效,极大地减小了合成和筛选的工作量,降低了筛选后亲和组分结构鉴定的难度。所建立的筛选策略已应用于流感病毒、严重急性呼吸道综合征(SARS)病毒亲和抑制剂的筛选和用于人β-干扰素测定的石英晶体微天平（QCM）生物传感器的构建,均取得了有意义的结果。     

Understanding ligand–protein interactions in affinity membrane chromatography for antibody purification

Affinity chromatography with Protein A beads has become the conventional unit operation for the primary capture of monoclonal antibodies. However, Protein A activated supports are expensive and ligand leakage is an issue to be considered. In addition, the limited production capabilities of the chromatographic process drive the research towards feasible alternatives. The use of synthetic ligands as Protein A substitutes has been considered in this work. Synthetic ligands, that mimic the interaction between Protein A and the constant fragment (Fc) of immunoglobulins, have been immobilized on cellulosic membrane supports. The resulting affinity membranes have been experimentally characterized with pure immunoglobulin G (IgG). The effects of the membrane support and of the spacer arm on the ligand–ligate interaction have been studied in detail. Experimental data have been compared with molecular dynamic simulations with the aim of better understanding the interaction mechanisms. Molecular dynamic simulations were performed in explicit water, modelling the membrane as a matrix of overlapped glucopyranose units. Electrostatic charges of the ligand and spacer were calculated through ab initio methods to complete the force field used to model the membrane. The simulations enabled to elucidate how the interactions of surface, spacer and ligand with IgG, contribute to the formation of the bond between protein and affinity membrane.     

Molecular modeling to rationalize ligand-support interactions in affinity chromatography

The development of rational design criteria for synthetic-ligand-based affinity chromatography requires a basic comprehension of all the factors influencing the binding capacity and selectivity of the stationary phase. In this work, molecular dynamics simulations are systematically used to investigate the impact of structural modifications of spacer and ligand on ligand-support interactions. The investigated ligands are characterized by a triazine core bi-functionalized with two amino acid side chains aimed at representing a range of hydrophobic/hydrophilic characters. As spacers both literature (1-2-diaminoethane and 1,4-substituted [1,2,3]-triazole) and speculative oligopeptidic molecules (Gly-[Ala]4-Gly, Gly-[Lys]4-Gly, and Gly-[Glu]4-Gly) have been considered to address the role of charges distribution, rigidity, and structural complexity. In this investigation, the spacer emerged as a key component: on the one hand, the choice of a proper spacer allows improving the hydrophilic character of the ligand-spacer adduct without compromising the structure of the affinity ligand, while on the other hand the use of structurally complex spacers induces spacer-support interactions that enhance the degree of solvation of the ligand regardless of its hydrophobic character. These findings suggest that the use of structured spacers could represent a viable pathway for tailoring the performances of affinity chromatography stationary phases.     

Design and synthesis of affinity ligands and relation of their structure with adsorption of proteins

Affinity chromatography has played an increasingly important role both in the pharmaceutical industry and academic research. In the present study, we report our preliminary investigation on the relationship between the affinity ligand structure and its adsorption to multi-protein samples. The structure of the ligands, including the size of the ring (cyclic group) and the length of the chain (linear group), has a great impact on the adsorption of ligands to proteins. Meanwhile, the functional groups that the ligands carry are also closely related to the adsorption of ligands to proteins. This research provides good guidance for the design and synthesis of affinity materials in affinity chromatography. It is also useful to other protein-ligand interaction-related research.     

Protein purification by aminosquarylium cyanine dye‐affinity chromatography

The most selective purification method for proteins and other biomolecules is affinity chromatography. This method is based on the unique biological‐based specificity of the biomolecule–ligand interaction and commonly uses biological ligands. However, these ligands may present some drawbacks, mainly because of their cost and lability. Dye‐affinity chromatography overcomes the limitations of biological ligands and is widely used owing to the low cost of synthetic dyes and to their resistance to biological and chemical degradation. In this work, immobilized aminosquarylium cyanine dyes are used in order to exploit affinity interactions with standard proteins such as lysozyme, α‐chymotrypsin and trypsin. These studies evaluate the affinity interactions occurring between the immobilized ligand and the different proteins, as a reflection of the sum of several molecular interactions, namely ionic, hydrophobic and van der Waals, spread throughout the structure, in a defined spatial manner. The results show the possibility of using an aminosquarylium cyanine dye bearing a N‐hexyl pendant chain, with a ligand density of 1.8 × 10^?2 mmol of dye/g of chromatographic support, to isolate lysozyme, α‐chymotrypsin and trypsin from a mixture. The application of a decreasing ammonium sulfate gradient resulted in the recovery of lysozyme in the flowthrough. On the other hand, α‐chymotrypsin and trypsin were retained, involving different interactions with the ligand. In conclusion, this study demonstrates the potential applicability of ligands such as aminosquarylium cyanine dyes for the separation and purification of proteins by affinity chromatography. Copyright © 2013 John Wiley & Sons, Ltd.     

Selective adsorption of poly-His tagged glutaryl acylase on tailor-made metal chelate supports.

A poly-His tag was fused in the glutaryl acylase (GA) from Acinetobacter sp. strain YS114 cloned in E. coli yielding a fully active enzyme. Biochemical analyses showed that the tag did not alter the maturation of the chimeric GA (poly-His GA) that undergoes a complex post-translational processing from an inactive monomeric precursor to the active heterodimeric enzyme. This enzyme has been used as a model to develop a novel and very simple procedure for one-step purification of poly-His proteins via immobilized metal-ion affinity chromatography on tailor-made supports. It was intended to improve the selectivity of adsorption of the target protein on tailor-made chelate supports instead of performing a selective desorption. The rate and extent of the adsorption of proteins from a crude extract from E. coli and of pure poly-His tagged GA on different metal chelate supports was studied. Up to 90% of proteins from E. coli were adsorbed on commercial chelate supports having a high density of ligands attached to the support through long spacer arms, while this adsorption becomes almost negligible when using low ligand densities, short spacer arms and Zn2+ or Co2+ as cations. On the contrary, poly-His GA adsorbs strongly enough on all supports. A strong affinity interaction between the poly-His tail and a single chelate moiety seems to be the responsible for the adsorption of poly-His GA. By contrast, multipoint weak interactions involving a number of chelate moieties seem to be mainly responsible for adsorption of natural proteins. By using tailor-made affinity supports, a very simple procedure for one-step purification of GA with minimal adsorption of host proteins could be performed. Up to 20 mg of GA were adsorbed on each ml of chelate support while most of accompanying proteins were hardly adsorbed on such supports. Following few washing steps, the target enzyme was finally recovered (80% yield) by elution with 50 mM imidazole with a very high increment of specific activity (up to a 120 purification factor).     

Affinity purification of metalloprotease from marine bacterium using immobilized metal affinity chromatography

In this study, an efficient affinity purification protocol for an alkaline metalloprotease from marine bacterium was developed using immobilized metal affinity chromatography. After screening and optimization of the affinity ligands and spacer arm lengths, Cu‐iminmodiacetic acid was chosen as the optimal affinity ligand, which was coupled to Sepharose 6B via a 14‐atom spacer arm. The absorption analysis of this medium revealed a desorption constant K_d of 21.5 μg/mL and a theoretical maximum absorption Q_max of 24.9 mg/g. Thanks to this affinity medium, the enzyme could be purified by only one affinity purification step with a purity of approximately 95% pure when analyzed by high‐performance liquid chromatography and reducing sodium dodecyl sulfate polyacrylamide gel electrophoresis. The recovery of the protease activity reached 74.6%, which is much higher than the value obtained by traditional protocols (8.9%). These results contribute to the industrial purifications and contribute a significant reference for the purification of other metalloproteases.     

Affinity of trypsin for amidine derivatives immobilized on dextran-coated silica supports.

The pancreas contains two very analogous enzymes: trypsin and chymotrypsin. These two enzymes are very similar in their physicochemical characteristics and are therefore quite difficult to separate by classical purification procedures. They constitute a good model for affinity chromatography. It was previously demonstrated that amidine derivatives are able to interact strongly and specifically with these serine proteases and are often used as ligand in affinity chromatography. To understand the trypsin interaction mechanism, we synthesized different amidines and immobilised them with or without spacer arm on silica beads previously coated by dextran substituted with a calculated amount of positively charged diethylaminoethyl functions, in order to minimize the non-specific interactions of silanol groups of the silica material. First the affinity constant and the adsorption capacity of these supports for trypsin were determined in batch procedures, then they were used in affinity chromatography. The effects of ionic strength, pH and competitive inhibitors on proteins desorption were also studied. Last, to demonstrate the importance of passivation, the chromatographic performances of dextran-coated silica phases and a commercial support grafted with the same amidine were compared.     

Fast Affinity Chromatography Using Small Particle Silica-Based Packing Materials

Summary

Affinity chromatography is one of the most powerful techniques for the purification of biologically active proteins available (for review see [1]). The ability of this method to purify proteins is based on highly specific, selective or characteristic interactions with immobilized ligands. Several advantages over traditional soft gel affinity supports have been observed with the use of small particle silica based materials for high performance affinity chromatography. These include greatly improved mass transfer properties which allow separations that are not always practical in the low performance mode, greatly reduced equilibration and isolation times, high available ligand densities, small elution volumes, excellent recovery of very small quantities of protein and high dynamic capacities. The criteria for developing a general, derivatizable, high performance support for high performance affinity chromatography are discussed. The step-by-step examination of these criteria and experimental evidence for determining parameters such as ligand density, non-specific adsorption and column life time for such a system are described. Chromatographic results are shown for preparative separations of (i) receptor proteins, (ii) antibodies and (iii) active enzymes.     

Drug screening of pharmaceutical discovery compounds by micro-size exclusion chromatography/mass spectrometry.

Micro-size exclusion chromatography coupled with capillary liquid chromatography (capLC) and mass spectrometry (MS) provides a rapid and simple approach to the preliminary screening of active ligands toward a specific target macromolecule. In this study, the effectiveness of this technique is demonstrated by a number of small molecule ligands with known binding affinities towards the protein target. All ligands were incubated together with a target protein under native conditions. Separation was then achieved by microcentrifugation where the high molecular weight (MW) compounds were selectively passed through the size-exclusion material. The retained low MW compounds were then recovered and analyzed by capLC/MS. The absence of the ligand indicated strong affinity towards the target, while ligand detection indicated inactivity. This assay demonstrated the drugs that were acting as strong inhibitors of Co-PDF from those showing to be comparatively inactive. The relative binding rank order of the drugs towards Co-PDF was also determined. The results were validated by a corresponding set of control experiments in which the target molecules were excluded from the process. In principle, high-throughput micro-size exclusion chromatography, coupled with capLC/MS, offers a powerful technique as a preliminary screen in determining both the strong binding affinity and the relative affinity rank ordering of ligands towards a specific target macromolecule, and is complementary with other analytical drug screening techniques.     

Development and screening of epoxy‐spacer‐phage cryogels for affinity chromatography: Enhancing the binding capacity

Macroporous epoxy cryogels can be used as an alternative for classical matrices in affinity chromatography. Due to the structural properties of cryogels, with pores of up to 100 μm, crude samples can be processed at high speed without previous manipulations such as clarification or centrifugation. Also, we previously used a peptide‐expressing M13 bacteriophage as an affinity ligand. These ligands show high specificity toward the target to be purified. Combination of both, leads to a relative cost‐effective one‐step chromatographic set‐up delivering a high purity sample (>95%), however, so far with limited capacity. To increase the binding capacity of the affinity columns, we now inserted spacers between the chromatographic matrix and the phage ligand. Both linear spacers, di‐amino‐alkanes (C₂–C₁₀), and branched polyethyleneimine spacers with different molecular weights (800 Da–10 kDa) were analyzed. Two types of peptide expressing phage ligands, a linear 15‐mer and a cyclic 6‐mer, were used for screening. Up to a tenfold increase in binding capacity was observed depending on the combination of phage ligand and spacer type.     

Calcium-modulated conformational affinity chromatography. Application to the purification of calmodulin and S100 proteins.

The purification of proteins by affinity chromatography is based on their highly specific interaction with an immobilized ligand followed by elution under conditions where their affinity towards the ligand is markedly reduced. Thus, a high-degree purification by a single chromatographic step is achieved. However, when several proteins in the crude mixture share affinity to a common immobilized ligand, they may not be resolved by affinity chromatography and subsequent "real" chromatographic purification steps may be required. It is shown that by using properly selected gradient elution conditions, the affinities of the various proteins towards the immobilized ligand may be gradually modulated and their separation may be achieved. This is exemplified by the isolation and separation of a group of Ca(2+)-activated proteins, Calmodulin, S100a and S100b, from bovine brain extract, using a melittin-Eupergit C affinity column which is developed with Ca(2+)-chelator gradients. As expected, separation of the three proteins into individual peaks, eluted in order of increasing affinity to the matrix, was obtained. Sigmoid selectivity curves calculated from the elution volumes under different elution conditions for each of the proteins were obtained, illustrating the chromatographic behaviour of the gradient affinity separation system.     

Molecular-scale neural nets: an approach to the self-assembly of molecular networks

It is suggested that molecular self-assembly techniques may be utilised in molecular scale electronic systems. Taking a neural network as an example, it is shown how the affinity of proteins for their complementary ligand could be used to form the molecular scale network essential to such a system. The approach is based on the polymerisation of the tetrameric proteins avidin and streptavidin using biotinylated ligands. By synthesising a range of aromatic bisbiotin ligands, some simple ground rules are established for ensuring complexation of protein and ligand. Based on these findings, a tetrabiotinylated tetrapyridylporphyrin salt (TBPP) was synthesised with a view to forming a two-dimensional protein polymer. Gel chromatography experiments and a UV-visible investigation of solutions containing the protein/TBPP mixture provide strong evidence for complex formation. The specific immobilisation of streptavidin to a monolayer of TBPP formed at the air-water interface is also demonstrated using fluorescence microscopy. Based on the promising results of these initial studies, a number of suggestions are made for the further exploitation of the techniques described herein.     

Purification of guanosine triphosphate cyclohydrolase I from Escherichia coli. The use of competitive inhibitors versus substrate as ligands in affinity chromatography

Different affinity chromatography ligands have been compared for the purification of guanosine triphosphate (GTP) cyclohydrolase I, an enzyme that catalyses the transformation of GTP into formate and dihydroneopterin triphosphate, the first metabolite in the biosynthetic pathway of the pterins. When this enzyme is purified by affinity chromatography on GTP-Sepharose a major fraction of the activity is lost and the yield of enzyme decreases as the amount of enzyme applied to the column decreases. The use of nucleotide competitive inhibitors (UTP and ATP) as ligands in the affinity column has shown that the extent of inactivation of the enzyme is related to the affinity of the enzyme for the ligand. Further, the extent of inactivation was reduced by reducing the length of the columns when using the same volume of GTP-Sepharose. Dihydrofolate-Sepharose gave consistently higher yields of GTP cyclohydrolase I regardless of the amount of enzyme applied, but several other proteins were also obtained. For a high purification of GTP cyclohydrolase I the best yield may be obtained with UTP as the affinity ligand and with the shortest length possible of the affinity column, and the purity of enzyme is comparable with that obtained with GTP-Sepharose.     

Tetravalent Pseudomonas aeruginosa Adhesion Lectin LecA Inhibitor for Enhanced Biofilm Inhibition

A potent divalent ligand of the Pseudomonas aeruginosa adhesion lectin LecA was elaborated into a tetravalent version. A polyethylene glycol (PEG) spacer was introduced to link two divalent galactosides. Each of the two divalent ligands contained a rigid spacer with a central phenyl group that is bridged by the PEG moiety. The resulting tetravalent ligand was found to bind LecA in the nanomolar range involving all of its sugar (sub)ligands. Analytical ultracentrifugation studies clearly showed that the tetravalent ligand was capable of aggregation the LecA tetramers in contrast to the divalent ligands. The aggregator behavior was found to be of importance in P. aeruginosa biofilm formation inhibition. Despite the weaker affinity it was a considerably better biofilm inhibitor with half inhibitory values around the 28 micromolar range.     

分离尿激酶的胍基型亲和色谱填料研究

尿激酶的精制一般使用亲和色谱法［’－‘ｊ．目前实际使用和文献报道最多的填料是在 ＳｅＰｈａｒｏｓｅ上键合对氨基苯眯（ｐ－ＡＢＺ）制得的［‘ｊ．这种填料分高效果较好,但机械强度不高,只能用于常压色谱,而且寿命较短．前文［‘１报道了用 ＳｅＰｈａｒｏｓｅ和聚甲基丙烯酸环氧丙酯微球为基质, Ｐ－ＡＢＺ为配基分离尿激酶的亲和色谱填料的对照研究．本文报道以含肥基的有机小分子为配基,ＳｅＰｈａｒｏｓｅ及两种聚甲基丙烯酸环氧丙酯微球为基质的分离尿激酶色谱填料的合成和性能试验．发现肥基己酸和精氨酸为配基的亲和色谱…     

Bovine lactoferrin purification from whey using Yellow HE‐4R as the chromatographic affinity ligand

The worldwide production of whey increases by around 186 million tons each year and it is generally considered as a waste, even when several whey proteins have important economic relevance. For its valorization, inexpensive ligands and integrated chromatography methods need to be developed for specific and low‐cost protein purification. Here, we describe a novel affinity process with the dye Yellow HE‐4R immobilized on Sepharose for bovine lactoferrin purification. This approach based on a low‐cost ligand showed an efficient performance for the recovery and purification of bovine lactoferrin directly from whey, with a yield of 71% and a purification factor of 61.