Protein A磁性纳米颗粒载体的制备及应用

本研究采用本课题组合成的表面氨基化磁性纳米微球,首先通过化学共价交联制备了葡萄球菌Protein A磁性纳米微球载体（SPA-MP）,并探讨了载体制备的优化条件。然后根据生物分子特异性亲合作用原理,在外加磁场的定向控制下,通过亲和吸附、清洗和解吸附等操作,探讨了SPA-MP载体在抗体分离纯化领域的应用可行性。载体制备优化实验结果显示,通过改变蛋白质浓度、交联剂浓度和交联剂活化时间可以制备不同表面密度的SPA-MP载体。300 μg SPA,2.5% (V/V)戊二醛浓度和3小时的活化时间可以获取表面密度高达35 mg SPA/g磁性纳米微球的载体。此外,应用结果显示每克SPA-MP磁性微球载体可以结合高达14 mg 的CD25抗体,同时可有效地分离纯化人抗血清样品中的IgG抗体。     

葡萄球菌蛋白A活性机制与现代应用

葡萄球菌蛋白A(staphylococcus protein A,SPA)是金黄色葡萄球菌(Staphylococcus aureus)细胞壁上的一种黏连蛋白.经过几十年的研究,由于其对免疫球蛋白IgG的亲和特性,SPA已作为一种工具在抗体的检测、纯化以及疾病诊断中得到了广泛应用.SPA衍生物的构建适应了现代生产的需要,改造后的SPA可以与多种报告分子相偶联,使得免疫诊断更加快速准确.本文综述了SPA基于其免疫学特性,在抗体纯化和现代免疫分析应用中的发展状况及前景.     

Improving the tolerance of a protein a analogue to repeated alkaline exposures using a bypass mutagenesis approach

Staphylococcal protein A (SPA) is a cell surface protein expressed by Staphylococcus aureus. It consists of five repetitive domains. The five SPA-domains show individual interaction to the Fc-fragment as well as certain Fab-fragments of immunoglobulin G (IgG) from most mammalian species. Due to the high affinity and selectivity of SPA, it has a widespread use as an affinity ligand for capture and purification of antibodies. One of the problems with proteinaceous affinity ligands in large-scale purification is their sensitivity to alkaline conditions. SPA however, is considered relatively stable to alkaline treatment. Nevertheless, it is desirable to further improve the stability in order to enable an SPA-based affinity medium to withstand even longer exposure to the harsh conditions associated with cleaning-in-place (CIP) procedures. For this purpose, a protein engineering strategy, which was used earlier for stabilization and consists of replacing the asparagine residues, is employed. Since Z in its "nonengineered" form already has a significant tolerance to alkaline treatment, small changes in stability due to the mutations are difficult to assess. Hence, in order to enable detection of improvements regarding the alkaline resistance of the Z domain, we chose to use a bypass mutagenesis strategy using a mutated variant Z(F30A) as a surrogate framework. Z(F30A) has earlier been shown to possess an affinity to IgG that is similar to the wild-type but also demonstrates decreased structural stability. Since the contribution of the different asparagine residues to the deactivation rate of a ligand is dependent on the environment and also the structural flexibility of the particular region, it is important to consider all sensitive amino acids one by one. The parental Z-domain contains eight asparagine residues, each with a different impact on the alkaline stability of the domain. By exchanging asparagine 23 for a threonine, we were able to increase the stability of the Z(F30A) domain in alkaline conditions. Also, when grafting the N23T mutation to the Z scaffold, we were able to detect an increased tolerance to alkaline treatment compared to the native Z molecule.     

Tandem affinity purification vectors for use in gram positive bacteria

Tandem affinity purification has become a valuable tool for the isolation of protein complexes. Here we describe the construction and use of a series of plasmid vectors for Gram positive bacteria. The vectors utilize the SPA tag as well as variants containing a 3C rather than the TEV protease site as 3C protease has been shown to work efficiently at the low temperatures (4 degrees C) used to isolate protein complexes. In addition, a further vector incorporates a GST moiety in place of the 3xFLAG of the SPA tag which provides an additional tagging option for situations where SPA binding may be inefficient. The vectors are all compatible with previously constructed fluorescent protein fusion vectors enabling construction of a suite of affinity and fluorescently tagged genes using a single PCR product.     

Molecules incorporating a benzothiazole core scaffold inhibit the N-myristoyltransferase of Plasmodium falciparum

Recombinant N-myristoyltransferase of Plasmodium falciparum (termed PfNMT) has been used in the development of a SPA (scintillation proximity assay) suitable for automation and high-throughput screening of inhibitors against this enzyme. The ability to use the SPA has been facilitated by development of an expression and purification system which yields considerably improved quantities of soluble active recombinant PfNMT compared with previous studies. Specifically, yields of pure protein have been increased from 12 microg x l(-1) to >400 microg x l(-1) by use of a synthetic gene with codon usage optimized for expression in an Escherichia coli host. Preliminary small-scale 'piggyback' inhibitor studies using the SPA have identified a family of related molecules containing a core benzothiazole scaffold with IC50 values <50 microM, which demonstrate selectivity over human NMT1. Two of these compounds, when tested against cultured parasites in vitro, reduced parasitaemia by >80% at a concentration of 10 microM.     

Sequential Peptide Affinity (SPA) system for the identification of mammalian and bacterial protein complexes

A vector system is described that combines reliable, very low level, regulated protein expression in human cells with two affinity purification tags (Sequential Peptide Affinity, or SPA, system). By avoiding overproduction of the target protein, this system allows for the efficient purification of natural protein complexes and their identification by mass spectrometry. We also present an adaptation of the SPA system for the efficient purification and identification of protein complexes in E. coli and, potentially, other bacteria.     

Anti-idiotypic protein domains selected from protein A-based affibody libraries

Three pairs of small protein domains showing binding behavior in analogy with anti-idiotypic antibodies have been selected using phage display technology. From an affibody protein library constructed by combinatorial variegation of the Fc binding surface of the 58 residue staphylococcal protein A (SPA)-derived domain Z, affibody variants have been selected to the parental SPA scaffold and to two earlier identified SPA-derived affibodies. One selected affibody (Z(SPA-1)) was shown to recognize each of the five domains of wild-type SPA with dissociation constants (K(D)) in the micromolar range. The binding of the Z(SPA-1) affibody to its parental structure was shown to involve the Fc binding site of SPA, while the Fab-binding site was not involved. Similarly, affibodies showing anti-idiotypic binding characteristics were also obtained when affibodies previously selected for binding to Taq DNA polymerase and human IgA, respectively, were used as targets for selections. The potential applications for these types of affinity pairs were exemplified by one-step protein recovery using affinity chromatography employing the specific interactions between the respective protein pair members. These experiments included the purification of the Z(SPA-1) affibody from a total Escherichia coli cell lysate using protein A-Sepharose, suggesting that this protein A/antiprotein A affinity pair could provide a basis for novel affinity gene fusion systems. The use of this type of small, robust, and easily expressed anti-idiotypic affibody pair for affinity technology applications, including self-assembled protein networks, is discussed.     

新型蛋白质配体-亲和体研究进展

亲和体(affibody)是一种衍生于葡萄球菌A蛋白B结构域的人工蛋白质分子．B结构域含58个氨基酸,形成3个α螺旋结构,分子质量约为6.5 ku．其中第一及第二螺旋中的13个特定位点的氨基酸对其结构无明显影响,这些位点可被随机突变形成理论上可与任何靶分子结合的亲和体文库．筛选该文库可获得能与某一靶分子特异结合的亲和体．亲和体与靶分子的结合特性与抗体相似,但与抗体相比具有一些独特的优势,如：通过体外筛选即可获得,以化学合成方法或原核表达即可大量制备,分子质量小、在生物体内组织穿透性强、血浆清除率高,理化稳定性好,可以通过交联或融合表达与标记分子(如荧光蛋白、生物素等)结合而不影响其与靶分子的结合能力．亲和体可作为抗体的替代品,用于蛋白质识别、分离及纯化、实验诊断、分子显像及靶向治疗．     

Intein-mediated protein purification of fusion proteins expressed under high-cell density conditions in E. coli

The intein-mediated purification system has the potential to significantly reduce the recovery costs of industrial recombinant proteins. The ability of inteins to catalyze a controllable peptide bond cleavage reaction can be used to separate a recombinant protein from its affinity tag during affinity purification. Inteins have been combined with a chitin-binding domain to serve as a self-cleaving affinity tag, facilitating highly selective capture of the fusion protein on an inexpensive substrate--chitin (IMPACT) system, New England Biolabs, Beverly, MA). This purification system has been used successfully at a lab scale in low cell density cultures, but has not been examined comprehensively under high-cell density conditions in defined medium. In this study, the intein-mediated purification of three commercially relevant proteins expressed under high-cell density conditions in E. coli was studied. Additionally, losses during the purification process were quantified. The data indicate that the intein fusion proteins expressed under high cell density fermentations were stable in vivo after induction for a significant duration, and the intein fusion proteins could undergo thiol or pH and temperature initiated cleavage reaction in vitro. Thus, the intein-mediated protein purification system potentially could be employed for the production of recombinant proteins at the industrial-scale.     

Preparation of Group I Introns for Biochemical Studies and Crystallization Assays by Native Affinity Purification

The study of functional RNAs of various sizes and structures requires efficient methods for their synthesis and purification. Here, 23 group I intron variants ranging in length from 246 to 341 nucleotides—some containing exons—were subjected to a native purification technique previously applied only to shorter RNAs (<160 nucleotides). For the RNAs containing both exons, we adjusted the original purification protocol to allow for purification of radiolabeled molecules. The resulting RNAs were used in folding assays on native gel electrophoresis and in self-splicing assays. The intron-only RNAs were subjected to the regular native purification scheme, assayed for folding and employed in crystallization screens. All RNAs that contained a 3′ overhang of one nucleotide were efficiently cleaved off from the support and were at least 90% pure after the non-denaturing purification. A representative subset of these RNAs was shown to be folded and self-splicing after purification. Additionally, crystals were grown for a 286 nucleotide long variant of the Clostridium botulinum intron. These results demonstrate the suitability of the native affinity purification method for the preparation of group I introns. We hope these findings will stimulate a broader application of this strategy to the preparation of other large RNA molecules.     

Camelid VHH affinity ligands enable separation of closely related biopharmaceuticals

Interest in new and diverse classes of molecules such as recombinant toxins, enzymes, and blood factors continues to grow for use a biotherapeutics. Compared to monoclonal antibodies, these novel drugs typically lack a commercially available affinity chromatography option, which leads to greater process complexity, longer development timelines, and poor platformability. To date, for both monoclonal antibodies and novel molecules, affinity chromatography has been mostly reserved for separation of process‐related impurities such as host cell proteins and DNA. Reports of affinity purification of closely related product variants and modified forms are much rarer. In this work we describe custom affinity chromatography development using camelid V_HH antibody fragments as "tunable" immunoaffinity ligands for separation of product‐related impurities. One example demonstrates high selectivity for a recombinant immunotoxin where no binding was observed for an undesired deamidated species. Also discussed is affinity purification of a coagulation factor through specific recognition of the gamma‐carboxylglutamic acid domain.     

双杂交和质谱技术在蛋白质-蛋白质相互作用研究中的应用

基因的功能是由蛋白质来执行的,而蛋白质要通过与其他生物分子相互作用来完成其各种生物功能。因此,如果能够快速做出蛋白质在不同时间、空间和不同环境中的相互作用图谱,就会帮助我们了解这些蛋白质的功能,进而了解许多生命活动的机制。目前,用于大规模研究蛋白质间相互作用的方法主要有酵母双杂交系统及其衍生系统、亲和纯化与质谱分析联用技术,前者用于研究蛋白分子间的两两相互作用,后者用于研究蛋白质复合物间的相互作用。本文主要阐述了酵母双杂交、细菌双杂交、哺乳动物细胞双杂交、亲和纯化与质谱联用技术在大规模蛋白质相互作用研究中的应用。     

Subunit composition of the purified dihydropyridine binding protein from skeletal muscle

The dihydropyridine (DHP) receptor from rabbit skeletal muscle has been characterized by affinity labeling and purification. Two procedures were used for purification: one that was a procedure modified from that of Curtis and Catterall (1984) and one that employed an anti alpha 1 monoclonal antibody (Mab) affinity column. In addition, both digitonin and CHAPS solubilizations were utilized with each purification technique. The major findings are as follows: (1) In contrast to the behavior in digitonin, neither the 52K (beta) nor the 140K (alpha 2) polypeptide quantitatively copurifies with the 170K (alpha 1) polypeptide when the purification is carried out in CHAPS. This has been shown by use of both wheat germ and monoclonal antibody columns. The digitonin-extracted receptor complex bound to the Mab affinity column loses alpha 2 and beta when the digitonin is replaced by CHAPS, and when the complex is bound to a WGA column, a CHAPS wash causes dissociation of alpha 1, beta, and gamma from alpha 2. Loss of binding of dihydropyridines occurs with the CHAPS wash but can be partially restored by the addition of the CHAPS wash to the material eluted from the column with N-acetylglucosamine. (2) Although both detergents solubilized greater than 80% of the polypeptides associated with the DHP binding site, the ability of these proteins to bind dihydropyridines is reduced more by CHAPS treatment than by digitonin treatment, raising the possibility that subunit interactions contribute to high-affinity binding. Alternatively, CHAPS may remove tightly bound lipids necessary for binding or cause irreversible denaturation of the binding site.(ABSTRACT TRUNCATED AT 250 WORDS)     

Protected amino acids as novel low-molecular-weight displacers in cation-exchange displacement chromatography

Although the ability to carry out simultaneous concentration and purification in a single displacement step has significant advantages for downstream processing of pharmaceuticals, a major impediment to the implementation of displacement chromatography has been the lack of suitable displacer compounds. An important recent advance in the state of the art of displacement chromatography has been the discovery that low-molecular-weight dendritic polymers can be successfully employed as displacers for protein purification in ion-exchange systems. In this article, protected amino acid esters (based on arginine and lysine) are shown to be useful displacers for protein purification in cation-exchange systems. A dynamic affinity plot is employed to evaluate the affinity of these low-molecular-weight compounds under dis-placement conditions. In contrast to large polyelectroyte displacers, the efficacy of these low-molecular-weight displacers was shown to be dependent on both the initial carrier salt concentration and the displacer concentration. In addition to the funcamental interest generated by low-molecular-weight displacers, it is likely that these displacers will have significant operatioal advantages as compared with large polyelectrolyte displacers. (c) 1995 John Wiley & Sons, Inc.     

A universally applicable process for preparing stoichiometrically 1:1 labelled functional proteins

A universally applicable labelling and purification process was established to prepare biologically active proteins with a stoichiometric 1:1 ratio of attached dye-label. The dye-label is linked to a specific DNA sequence, which acts as a barcode-like tag for affinity purification. The DNA-dye tag is covalently bound to the target protein, which is present in excess to assure the binding of not more than one dye per molecule. Affinity purification occurs at magnetic beads that are functionalized with oligonucleotides that are complementary to the DNA-tag of the labelled proteins but for one or two mismatches. Washing removes all unbound, unlabelled molecules. The labelled protein is subsequently released by the addition of a fully complementary oligonucleotide. This process allows a gentle purification of a protein fraction that has exactly one label attached to each molecule under conditions that preserve protein structure.     

Human IgA and IgG F(ab')2 that bind to staphylococcal protein A belong to the VHIII subgroup

Staphylococcal protein A (SPA) is a bacterial membrane protein that possesses, in addition to its Fc gamma-binding activity, a distinct specificity for the Fab region of some IgM, IgA, IgG, and IgE. The Fab site that binds to SPA has been localized to the V region of the Ig H chain. In a previous study of human monoclonal and polyclonal IgM, we demonstrated that binding to SPA was highly restricted to molecules of the VHIII subgroup, and that nearly all VHIII IgM were able to bind SPA. The present study examines the VH composition of SPA-binding and SPA-nonbinding fractions of purified human polyclonal IgA, and IgG F(ab')2 fragments. We found that 22% of the IgA and 15% of the IgG F(ab')2 bound to SPA-agarose. Analysis with VH subgroup-specific antisera indicated that the SPA-binding fraction of IgA was dominated by the VHIII subgroup, and the SPA-binding fraction of IgG F(ab')2 contained only VHIII molecules. Furthermore, substantial portions of the total VHIII protein in IgA and in IgG F(ab')2 bound to SPA. We conclude that Fab binding to SPA is both restricted to and highly prevalent among human VHIII molecules, regardless of Ig class. These results suggest that protein A is an Ig superantigen.     

Src homology-2 domain binding assays by scintillation proximity and surface plasmon resonance

Various SH2 competitive binding assays, based on different techniques, have been described in the literature to identify and characterize SH2 ligands. The consideration that most reported methods show experimental limitations associated with assay parameters has prompted us to base our Src-SH2 inhibitor discovery program on the use of two different assays. In this study, two conceptually different biochemical methods designed to discover Src-SH2 inhibitors, respectively scintillation proximity assay (SPA) and surface plasmon resonance (SPR), have been evaluated and compared. For its high sensitivity and adaptability to automation SPA was chosen for high capacity screening (primary screen), whereas SPR was used for hits confirmation (secondary screening). However with the drastic improvement of inhibitor affinities, the limit of sensitivity was rapidly reached for the SPR assay based on the canonical pYEEI ligand. The substitution of the natural, monophosphorylated peptide ligand with a triphosphorylated peptide has allowed us to remarkably increase its sensitivity, so that molecules with nanomolar affinities could be easily differentiated in terms of IC(50) ranking. Such a new, improved SPR assay can be of great interest for the study of high affinity ligands of different SH2-based drug targets.     

Characterization of the rat liver glucocorticoid receptor purified by DNA-cellulose and ligand affinity chromatography

Two rapid and high yield purification methods for the rat liver glucocorticoid receptor based on differential DNA affinity (method A) and ligand affinity (method B) chromatography are described. In method A, the amount of receptor in rat liver cytosol that can be activated and subsequently eluted from a DNA-cellulose column has been increased to 80% by introducing a second heat activation step. Using this method, 1.5 nmol of 25% pure glucocorticoid receptor can be routinely obtained per day from 15-20 rat livers. Method B yields about 2.2 nmol of 60% pure receptor with an overall yield of congruent to 60%. The quality of these purifications has been controlled by affinity labeling. In each case, more than 95% of purified binding activity represented the intact 92,000 +/- 400-Da glucocorticoid receptor polypeptide as shown by sodium dodecyl sulfate-gel electrophoresis and fluorography. No difference in the labeling pattern was observed using either [3H]triamcinolone acetonide (photoaffinity labeling) or [3H]dexamethasone 21-mesylate (electrophilic labeling). The electrophilic labeling step was performed in the cytosol prior to purification by method A to compare the labeled components thus purified with those obtained when the photoaffinity labeling was performed after the purification. Using this approach, distinct breakdown products of the glucocorticoid receptor were revealed, co-purifying during DNA affinity chromatography. Cross-linked receptor obtained by method A has been further purified to homogeneity by preparative sodium dodecyl sulfate-gel electrophoresis and successfully used as immunogen to raise glucocorticoid receptor antibodies in rabbits. These antibodies raised against glucocorticoid receptor, as well as those previously obtained using affinity chromatography-purified receptor, react with the receptor molecules irrespective of their method of purification. Glucocorticoid receptors purified by methods A and B have been analyzed for specific DNA-binding properties by the nitrocellulose filter binding assay.     

Differential IgG-binding characteristics of staphylococcal protein A, streptococcal protein G, and a chimeric protein AG

Various Gram-positive bacteria express different types of IgG-binding receptors, each of which displaying certain unique binding properties. To evaluate specificity and avidity aspects of the differential binding pattern, a set of competitive binding assays was employed, by using staphylococcal protein A (SPA), streptococcal protein G (SPG), and a chimeric protein AG. These receptors were analyzed, in a reciprocal fashion, for binding and inhibition of binding to a selected panel of polyclonal and monoclonal Ig. Results of the study reveal that a majority of the determinants on human and bovine IgG, recognized by SPA and SPG, are either coextensive or closely overlapping. Accordingly, a minor portion of the determinants appear to be unique in the sense that a particular determinant(s) is selectively identified by one of the two receptors. Binding assays involving purified Fc fragments from human IgG, suggest that SPG shows exclusive specificity for an Fab region determinant(s) not recognized by SPA, whereas the Fc determinants for SPA and SPG are identical or overlapping. Furthermore, one of the IgG subclasses of bovine origin appears to be seen by the SPG receptor only. The competition study also demonstrates that the novel chimeric protein AG receptor shows higher or equal avidity for variants of human IgG molecules compared to the best of its parental constituents. It can thus be deduced that chimeric receptors might be useful as optimized tools for immunologic applications.     

Purification of oligonucleotides by high affinity, low molecular weight displacers

High affinity, low molecular weight anionic displacers were successfully employed for the purification of antisense oligonucleotides. Several important structural characteristics were identified that contribute to the affinity of low molecular weight displacers to a hydrophilized polystyrene divinyl benzene anion exchanger. Sulfonic acid groups were found to possess higher affinity than carboxylic acid and phosphate functionalities, and nonspecific interactions (particularly hydrophobic interactions) were shown to play a major role in the retention process on this stationary phase material. Using this information, two high affinity, low molecular weight displacers were identified. These molecules are relatively inexpensive organic dyes that possess multiple sulfonic acid moieties, as well as aromatic functionalities, which increase nonspecific interactions with the stationary phase. These high affinity displacers, which can be readily detected, were then employed to displace several strongly retained antisense oligonucleotides that could not be displaced by previously established low molecular weight displacers. The displacement process resulted in very high purities of the antisense oligonucleotides. The results presented in this paper are significant in that they demonstrate that low molecular weight displacers for ion-exchange chromatography can possess equal to or greater affinities than their higher molecular weight counterparts, when nonspecific interactions with the stationary phase are exploited. In addition, the results illustrate the high resolutions possible with displacement chromatography and demonstrate an attractive technology for the process scale purification of oligonucleotides.