亲和-膜过滤技术及其应用

亲和-膜过滤技术是一项兼有亲和与膜分离优点的新型分离技术。介绍了亲和-膜过滤技术体系、亲和载体的制备及与目标分子的结合方式,综述了其在生物分离工程中的应用状况,指出今后研究工作的重点是亲和载体的开发及相关过程理论和技术的完善。     

Conversion of Low‐Affinity Peptides to High‐Affinity Peptide Binders by Using a β‐Hairpin Scaffold‐Assisted Approach

Affinity maturation of protein‐targeting peptides is generally accomplished by homo‐ or heterodimerization of known peptides. However, applying a heterodimerization approach is difficult because it is not clear a priori what length or type of linker is required for cooperative binding to a target. Thus, an efficient and simple affinity maturation method for converting low‐affinity peptides into high‐affinity peptides would clearly be advantageous for advancing peptide‐based therapeutics. Here, we describe the development of a novel affinity maturation method based on a robust β‐hairpin scaffold and combinatorial phage‐display technology. With this strategy, we were able to increase the affinity of existing peptides by more than four orders of magnitude. Taken together, our data demonstrate that this scaffold‐assisted approach is highly efficient and effective in generating high‐affinity peptides from their low‐affinity counterparts.     

亲和胶质气体泡沫的制备及其对胰蛋白酶的吸附分离特性

Triton X-114, an non-ionic surfactant, was modified with the affinity ligand of trypsin, paminobenzamidine (PAB) and the affinity surfactant (PAB-TX) was synthesized. Then, the affinity surfactant was used to prepare affinity-based colloidal gas aphrons (CGA). The stability of the affinity CGA was investigated at different temperatures and compared with that of the CGA prepared from Triton X-114. Compared with the CGA from Triton X-114, the affinity CGA showed high selective adsorption property for trypsin. In the separation of a protein mixture, recovery yield higher than 74% were achieved for trypsin and the separation factor reached over 1.5. The results showed that the affinity CGA possessed promising selectivity for separating trypsin from a protein mixture.     

亲和过滤技术研究进展

本文介绍了亲和过滤技术原理及其在蛋白质分离纯化中的应用研究现状，总结了建立样和过滤体系的一般方法，提出了多级亲和过滤过程研究的建议。     

Preparation of a heterogeneous hollow‐fiber affinity membrane modified with a mercapto chelating resin

A high‐quality, heterogeneous hollow‐fiber affinity membranes modified with mercapto was prepared through phase separation with blends of a chelating resin and polysulfone as membrane materials, poly(ethylene glycol) as an additive, N,N‐dimethylacetamide as a solvent, and water as an extraction solvent. The effects of the blending ratio and chelating resin grain size on the structure of the hollow‐fiber affinity membrane were studied. The effects of the composition of the spin‐cast solution and process parameters of dry–wet spinning on the structure of the heterogeneous hollow‐fiber affinity membrane were investigated. The pore size, porosity, and water flux of the hollow‐fiber affinity membrane all decreased with an increase in the additive content, bore liquid, and dry‐spinning distance. With an increase in the extrusion volume outflow, the external diameter, wall thickness, and porosity of the hollow‐fiber affinity membrane all increased, but the pore size and water flux of the hollow‐fiber affinity membrane decreased. It was also found that the effects of the internal coagulant composition and external coagulant composition on the structure of the heterogeneous hollow‐fiber affinity membrane were different. The experimental results showed that thermal drawing could increase the mechanical properties of the heterogeneous hollow‐fiber affinity membrane and decrease the pore size, porosity, and water flux of the heterogeneous hollow‐fiber affinity membrane, and the thermal treatment could increase the homogeneity and stability of the structure of the heterogeneous hollow‐fiber affinity membrane. © 2007 Wiley Periodicals, Inc. J Appl Polym Sci 2007     

亲和膜手性拆分技术及手性拆分固膜

阐述了亲和膜拆分技术,包括亲和超滤、纳滤、电渗析、渗析及渗透汽化等在手性拆分领域的应用,对基于对映体间亲和性差异的手性选择膜及基于"形状记忆"的分子印迹拆分膜的最新进展进行了综述,并就今后的发展方向提出建议。     

THE BINDING BEHAVIOUR OF IMMOBILISED LOW MOLECULAR WEIGHT LIGAND WITH PEPTIDES IN BIOSENSOR-BASED SYSTEM

In this study a hovel metal ion affinity ligand was immobility onto the sensor chip. Three poly-histidine peptides were used to study the interaction of tile peptides and the immobilised metal ion affinity ligand via biosensor system . The results obtained in this study indicate that the affinity of immobilised Ni(Ⅱ) ion affinity ligand for these peptides appear to be related to the arrangement of the histidine residues in the peptides. This study first documents the application of biosensor technique for paptide screening.     

氟虫腈为配基的亲和介质制备及鱼类GABA受体纯化

以琼脂糖凝胶(Sepharose)CL-6B为载体,衍生化的氟虫腈为亲和配基,制备亲和层析介质,对其进行FT-IR和XPS表征。用制备的亲和层析介质分离鱼类脑组织中的GABA(γ-氨基丁酸)受体,研究分离蛋白质的效率。结果表明,成功将氟虫腈作为配体偶联到亲和介质上,偶联量为36.68μmol/g胶;SDS-聚丙烯酰胺凝胶电泳(SDS-PAGE)显示两条蛋白质条带,其相对分子质量约为44 kD和55 kD。     

High affinity IgG binding by FcgammaRI (CD64) is modulated by two distinct IgSF domains and the transmembrane domain of the receptor

The high affinity IgG receptor, FcgammaRI, is comprised of three immunoglobulin superfamily (IgSF) domains (EC1, EC2 and EC3), a single transmembrane spanning region, and a short cytoplasmic tail. We have shown a role for three separate domains of FcgammaRI in the high affinity binding of IgG. Affinity measurements of chimeric FcgammaRs in which EC1 and EC2 of FcgammaRI have been replaced with the homologous EC1 and/or EC2 domains of the low affinity IgG receptor, FcgammaRII indicate that both EC2 and EC3 are essential for high affinity binding of monomeric IgG. Identification of EC3 from FcgammaRI as the binding site for the monoclonal antibody 10.1, which blocks IgG binding, provides further evidence for the role of this domain in binding. In addition, we have found that the affinity of FcgammaRI is increased threefold when co-expressed with its accessory molecule, gamma-chain. Affinity measurements of further chimeras indicates that the transmembrane domain of FcgammaRI has a negative influence upon the affinity of the receptor. To account for these observations, we propose that receptor dimerization is required for maximal affinity of FcgammaRI. Dimerization may serve as the mechanism by which IgG binding triggers several FcgammaRI-mediated events.      

Chemically prepared hevein domains: effect of C-terminal truncation and the mutagenesis of aromatic residues on the affinity for chitin

Chemically prepared hevein domains (HDs), N-terminal domainof an antifungal protein from Nicotiana tabacum (CBP20-N) andan antimicrobial peptide from Amaranthus caudatus (Ac-AMP2),were examined for their affinity for chitin, a ß-1,4-linkedpolymer of N-acetylglucosamine. An intact binding domain, CBP20-N,showed a higher affinity than a C-terminal truncated domain,Ac-AMP2. The formation of a pyroglutamate residue from N-terminalGln of CBP20-N increased the affinity. The single replacementof any aromatic residue of Ac-AMP2 with Ala resulted in a significantreduction in affinity, suggesting the importance of the completeset of three aromatic residues in the ligand binding site. Themutations of Phe18 of Ac-AMP2 to the residues with larger aromaticrings, i.e. Trp, ß-(1-naphthyl)alanine or ß-(2-naphthyl)alanine,enhanced the affinity, whereas the mutation of Tyr20 to Trpreduced the affinity. The affinity of an HD for chitin mightbe improved by adjusting the size and substituent group of stackingaromatic rings.     

EQUILIBRIUM ANALYSIS OF SYNTHESIS GAS WITH COMPOSITION-DEPENDENT DEGREES OF FREEDOM

An analysis is presented of the equilibrium composition of syngas mixtures parametrized in terms of the syngas ratio and the hydrogen mole fraction, for fixed values of pressure and temperature. Conditions for the existence of single or multiple carbon-free solutions are determined by analysis of the carbon affinity equation, formulated by the method of atomic potentials. The system is characterized by two curves describing the maximum possible mole fraction of hydrogen, and the mole fraction of hydrogen at unit carbon affinity. These curves are tangent to each other at a single contact point. The unit carbon affinity curve consists of two branches, corresponding either to the higher or to the lower carbon affinity root becoming equal to one. These branches join together at the contact point of the maximum bounding and unit carbon affinity curves. The syngas ratio at the contact point is pressure-independent and is expressed as a function of temperature. A single carbon-free solution is obtained in the region below both branches of the unit carbon affinity curve. It is shown, however, that two distinct carbon-free solutions can be constructed in the region enclosed between the maximum bounding curve and the right branch of the unit carbon affinity curve.     

AK-Score: Accurate Protein-Ligand Binding Affinity Prediction Using an Ensemble of 3D-Convolutional Neural Networks

Accurate prediction of the binding affinity of a protein-ligand complex is essential for efficient and successful rational drug design. Therefore, many binding affinity prediction methods have been developed. In recent years, since deep learning technology has become powerful, it is also implemented to predict affinity. In this work, a new neural network model that predicts the binding affinity of a protein-ligand complex structure is developed. Our model predicts the binding affinity of a complex using the ensemble of multiple independently trained networks that consist of multiple channels of 3-D convolutional neural network layers. Our model was trained using the 3772 protein-ligand complexes from the refined set of the PDBbind-2016 database and tested using the core set of 285 complexes. The benchmark results show that the Pearson correlation coefficient between the predicted binding affinities by our model and the experimental data is 0.827, which is higher than the state-of-the-art binding affinity prediction scoring functions. Additionally, our method ranks the relative binding affinities of possible multiple binders of a protein quite accurately, comparable to the other scoring functions. Last, we measured which structural information is critical for predicting binding affinity and found that the complementarity between the protein and ligand is most important.     

Model-based mutagenesis to improve the enantioselective fractionation properties of an antibody

The binding affinity and specificity of recombinant antibodiescan be modified by site-directed mutagenesis. Here we have usedmolecular modelling of the variable domains of an enantiospecificantibody fragment to fine-tune its affinity so it is more suitablefor the fractionation of the drug enantiomers. We have shownearlier that the Fab fragment of this antibody specificallyrecognizes one enantiomer from the racemic mixture of a medicaldrug and that it can be used for the fractionation of theseenantiomers by affinity chromatography. However, the affinitywas unnecessarily high, requiring harsh elution conditions torelease the bound enantiomer. Thus, the continuous use of theantibody affinity columns was impossible. We made a homologymodel of the antibody and designed mutations to the antigen-bindingsite to decrease the affinity. Four out of five point mutationsshowed decreased affinity for the hapten. Two of the mutationswere also combined to construct a double mutant. The affinitycolumns made using one of the single mutants with lowered affinityand the double mutant were capable of multiple rounds of enantioseparation. Received April 23, 2003; revised September 25, 2003;; accepted October 21, 2003     

Affinity proteins and their generation

Engineered affinity proteins have, together with antibodies and antibody derivatives, become indispensable tools in many areas of life science and with an increasing number of applications. The need for high‐throughput methods for generation of these different affinity proteins is evident. Today, combinatorial protein engineering is the most successful strategy to generate novel affinity proteins of non‐immunoglobulin origin. In this approach, high‐complexity combinatorial libraries are constructed from which affinity proteins are isolated using appropriate selection methods, thus circumventing the need for detailed knowledge of the protein structure and the binding mechanism that is necessary in more rational approaches. Since the introduction of the phage display technology, several alternative selection systems have been developed for this purpose. This review presents briefly some of the more commonly used affinity proteins, and gives an overview of the different methods and challenges related to the generation of library diversity and the selection methods available for the isolation of affinity proteins with desired properties. Copyright © 2012 Society of Chemical Industry     

Automated production scale affinity purification of monoclonal antibodies

This paper describes the use of automation to improve the efficiency of affinity chromatography as a production technique. The use of automation in development of affinity processes is described together with an analysis of the benefits of automating production systems. The paper considers what factors are important in achieving an efficient affinity process on a production scale. The discussion is supported with data illustrating the use of immobilised Protein A to purify large quantities of monoclonal antibody from cell culture. The paper concludes with a brief discussion of possible future developments and extensions of automation in affinity processes.     

Preparation of highly magnetic chitosan particles and their use for affinity purification of enzymes

A novel protocol for preparation of highly magnetic chitosan particles, with a coercive force as high as 3500 Oe, has been developed. The surface of the particles was functionalized with aldehyde groups to facilitate the attachment of affinity ligands. The optimum conditions for the preparation of highly magnetic chitosan particles and immobilization of trypsin on magnetic particles were obtained; these particles were then used for affinity purification of aprotinin, and the conditions of affinity purification are discussed in detail. The proposed method was successfully applied to the affinity purification of aprotinin. Copyright © 2003 Society of Chemical Industry     

Improving Elution and Displacement Ion-Exchange Chromatography by Adjusting Eluent and Displacer Affinities

Abstract

Multicomponent interference in ion-exchange chromatography is examined in this paper. A multicomponent chromatography theory is used to analyze the effect of eluent affinity in elution chromatography, and displacer and presaturant affinity in displacement chromatography. The eluent should have an affinity between those of the feed components and near the strongest affinity species. In displacement chromatography, the displacer and presaturant affinities should bracket those of the desired products as closely as possible. Displacement chromatography produces pure product peaks, although the feed throughput is larger for elution chromatography. These conclusions are also valid for adsorption chromatography.     

多糖类亲和分离材料的研究进展

亲和分离技术的核心是设计亲和分离材料,基于分子识别与亲和机制,针对多糖类高分子化合物的结构特点,探讨了其材料的设计原则,并综述了国内外多糖类亲和分离材料的制备技术及其应用研究进展。在此基础上提出了进一步改进多糖类高分子化合物制备技术的措施,并展望了此类材料的应用前景。     

Investigation of the `switch-epitope' concept with random peptide libraries displayed as thioredoxin loop fusions

The `FLITRX' random peptide library, consisting of dodecamerloop peptides displayed on a thioredoxin-flagellin scaffoldon Escherichia coli, was used to select peptide sequences withaffinity for a monoclonal antibody. These peptides were furtherscreened for pH- and metal-sensitive antibody binding. Severalzinc-sensitive peptides were identified, termed `switch epitopes'.A soluble, monomeric thioredoxin loop (`Trxloop') insertionanalog of a FLITRX switch epitope was constructed and its antibodybinding properties were characterized by Western blots. Zinc-dependentantibody recognition was maintained in the Trxloop protein althoughthe apparent antibody affinity was lower. This Trxloop proteinbound to an immobilized metal affinity chromatography matrix,similar to a `histidine-patch' thioredoxin variant, and wasreversibly precipitated by 1 mM Zn²⁺ or Cu²⁺ ions. Residuesimportant for zinc and antibody binding were determined by site-directedmutagenesis. The Trxloop antibody affinity was increased bysaturation mutagenesis. Biotinylated Trxloop (`Biotrxloop')variants of the original and improved affinity Trxloop proteinswere constructed and characterized by surface plasmon resonancemeasurements. Increased antibody affinity was partially dueto a slower antibody desorption rate, although the relativeadsorption rates were dependent on the amount of immobilizedBiotrxloop protein, indicating an influence of avidity on theapparent affinity.     

No Major Role for Binding by Salivary Proteins as a Defense Against Dietary Tannins in Mediterranean Goats

We investigated whether Mediterranean goats use salivary tannin-binding proteins to cope with tannin-rich forages by determining the affinity of salivary or parotid gland proteins for tannic acid or quebracho tannin. Mixed saliva, sampled from the oral cavity, or parotid gland contents were compared to the intermediate affinity protein bovine serum albumin with a competitive binding assay. Goats that consume tannin-rich browse (Damascus) and goats that tend to avoid tannins (Mamber) were sequentially fed high (Pistacia lentiscus L.), low (vetch hay), or zero (wheat hay) tannin forages. Affinity of salivary proteins for tannins did not differ between goat breeds and did not respond to presence or absence of tannins in the diet. Proteins in mixed saliva had slightly higher affinity for tannins than those in parotid saliva, but neither source contained proteins with higher affinity for tannins than bovine serum albumin. Similarly, 3 months of browsing in a tannin-rich environment had little effect on the affinity of salivary proteins for tannin in adult goats of either breed. We sampled mixed saliva from young kids before they consumed forage and after 3 months of foraging in a tannin-rich environment. Before foraging, the saliva of Mamber kids had higher affinity for tannic acid (but not quebracho tannin) than the saliva of Damascus kids, but there was no difference after 3 months of exposure to tannin-rich browse, and the affinity of the proteins was always similar to the affinity of bovine serum albumin. Our results suggest there is not a major role for salivary tannin-binding proteins in goats. Different tendencies of goat breeds to consume tannin-rich browse does not appear be related to differences in salivary tannin-binding proteins.