Improved purification of recombinant adenoviral vector by metal affinity membrane chromatography

The increasing importance of adenoviral vectors for gene therapy clinical trials necessitates the development of processes suitable for large-scale and commercial production of adenovirus. Here, we evaluated a novel purification process combining an anion-exchange chromatography and an immobilized metal affinity membrane chromatography for the purification of recombinant adenovirus. Adenovirus was initially purified from clarified infectious lysate by anion-exchange chromatography using Q Sepharose XL resin and further polished using a Sartobind IDA membrane unit charged with Zn²⁺ ions as affinity ligands. The metal affinity membrane chromatography efficiently removed residual host cell impurities that co-eluted with adenovirus during the previous anion-exchange chromatography step. The metal affinity membrane chromatography also separated defective adenovirus particles from the infectious adenovirus fraction. Furthermore, the metal affinity membrane chromatography showed an improved yield, when compared with a conventional bead-based metal affinity chromatography. The purity and specific activity of the adenovirus prepared using this two-step chromatography was comparable to those of adenovirus produced by the conventional CsCl density centrifugation. Therefore, our data provide an improved method for the purification of adenoviral vectors for clinical applications.     

免疫亲和层析法纯化棕尾别麻蝇(Sarcophagaperegrina)幼虫血淋巴凝集素

 报道了利用免疫亲和层析法纯化棕尾别麻蝇幼虫血淋巴凝集素的结果．哺乳动物红细胞能够特异地吸附凝集素．用兔红细胞与麻蝇幼虫血淋巴凝集素形成的复合体免疫供血家兔,得到麻蝇幼虫血淋巴凝集素的抗体．再利用抗体制备亲和吸附柱,通过免疫亲和层析一次性纯化了麻蝇幼虫血淋巴凝集素． Ｓ Ｄ Ｓ Ｐ Ａ Ｇ Ｅ结果显示,该凝集素的分子量约为７３ ｋ Ｄ．这一结果,与用对麻蝇幼虫血淋巴凝集素有抑制作用的糖蛋白—胎球蛋白和甲状腺球蛋白为配基,亲和层析纯化的结果完全相同,表明用这种免疫亲和层析法纯化凝集素是可行的．为不清楚专一性识别糖或专一性识别糖不典型,难于用普通亲和层析纯化的凝集素,提供了一种有效的纯化方法．     

Review: cyclodextrins and their interaction with amylolytic enzymes

This review is concerned with inhibition of amylases by cyclodextrins (cyclic maltooligosaccharides), the interaction that occurs between amylases and cyclodextrins and the application of cyclodextrin affinity chromatography in the purification of amylases. In many cases, amylases that are competitively inhibited by cyclodextrins can be purified by cyclodextrin affinity chromatography with the cyclodextrins interacting with the active site on such enzymes. Interestingly amylases that are not competitively inhibited by cyclodextrins may also be purified by cyclodextrin affinity chromatography. Therefore, cyclodextrin affinity chromatography can function in the purification of such amylolytic enzymes with the interaction occurring at a site removed from the active site. In such cases it appears that the cyclodextrin is interacting with an affinity site or binding site that is present on some amylolytic enzymes. It seems that certain similarities occur among the binding sites of such enzymes. Literature concerning amylases, and their subsequent purification using cyclodextrin affinity chromatography is reviewed and the fundamental basis of the interaction of the cyclodextrin with amylolytic enzymes is discussed here.     

The use of dyes in protein purification.

The role of the matrix, ligand and linking mechanism in affinity chromatography is discussed, special emphasis being placed on the use of dyestuff molecules as ligands. Current knowledge of dye-protein interactions is outlined and problems arising from the use of conventional textile dyes as ligands are considered. Work on the synthesis of novel dye-like molecules designed specifically for affinity chromatography is reviewed. This is seen as leading to the development of improved affinity systems capable of advancing the utility of affinity chromatography in protein purification.     

Separation of the high affinity insulin-like growth factor I receptor from low affinity binding sites by affinity chromatography

We have identified high and low affinity insulin-like growth factor I (IGF I)-binding sites with mean dissociation constants of 0.37 and 6.25 nM, respectively, in solubilized placental membranes. We have separated these sites and purified the high affinity IGF I receptor 1,300-fold, with an overall yield of 9.9%, using wheat germ agglutinin-Sepharose chromatography, insulin affinity chromatography, and IGF I affinity chromatography. The Scatchard plot of IGF I binding to the high affinity receptor is linear, suggesting the purification of a single homogeneous class of binding sites. Insulin is two orders of magnitude less effective than IGF I in competitively inhibiting IGF I binding to this receptor. The high affinity IGF I receptor is composed of alpha and beta subunits with apparent molecular weights of 135,500 and 96,200, respectively. IGF I at concentrations of greater than or equal to 50 ng/ml stimulates autophosphorylation of the beta subunit of the purified high affinity receptor 4.6-fold. Low affinity IGF I-binding sites run through the IGF I affinity column or are eluted from the insulin affinity column. The separation of IGF I receptors with different binding affinities by sequential affinity chromatography will make it possible to examine directly the determinants of receptor affinity.     

Applications of affinity chromatography in proteomics

Affinity chromatography is a powerful protein separation method that is based on the specific interaction between immobilized ligands and target proteins. Peptides can also be separated effectively by affinity chromatography through the use of peptide-specific ligands. Both two-dimensional electrophoresis (2-DE)- and non-2-DE-based proteomic approaches benefit from the application of affinity chromatography. Before protein separation by 2-DE, affinity separation is used primarily for preconcentration and pretreatment of samples. Those applications entail the removal of one protein or a class of proteins that might interfere with 2-DE resolution, the concentration of low-abundance proteins to enable them to be visualized in the gel, and the classification of total protein into two or more groups for further separation by gel electrophoresis. Non-2-DE-based approaches have extensively employed affinity chromatography to reduce the complexity of protein and peptide mixtures. Prior to mass spectrometry (MS), preconcentration and capture of specific proteins or peptides to enhance sensitivity can be accomplished by using affinity adsorption. Affinity purification of protein complexes followed by identification of proteins by MS serves as a powerful tool for generating a map of protein-protein interactions and cellular locations of complexes. Affinity chromatography of peptide mixtures, coupled with mass spectrometry, provides a tool for the study of protein posttranslational modification (PTM) sites and quantitative proteomics. Quantitation of proteomes is possible via the use of isotope-coded affinity tags and isolation of proteolytic peptides by affinity chromatography. An emerging area of proteomics technology development is miniaturization. Affinity chromatography is becoming more widely used for exploring PTM and protein-protein interactions, especially with a view toward developing new general tag systems and strategies of chemical derivatization on peptides for affinity selection. More applications of affinity-based purification can be expected, including increasing the resolution in 2-DE, improving the sensitivity of MS quantification, and incorporating purification as part of multidimensional liquid chromatography experiments.     

Influence of the nature of coupling agents on insulin adsorption on supports grafted with sialic acid for high-performance affinity chromatography

Porous silica exhibits excellent mechanical properties for use as a stationary phase for high-performance liquid chromatography. However, negative surface charges make it unusable in its native state. For this reason, silica beads are coated with dextran polymers carrying a calculated amount of diethylaminoethyl groups. Both the minimization of non-specific interactions and the hydrophilic character of such supports allow their functionalization with biospecific ligands and finally their use in high-performance affinity chromatography of biological products. The use of these modified supports in high-performance affinity chromatography requires a better understanding of various characteristics of stationary phases. For this purpose, several techniques were utilized, in particular, size-exclusion chromatography and adsorption of radiolabelled albumin. These methods provided complementary information on the structure of these supports. Coated silica-based supports were functionalized with sialic acid by means of different coupling agents. The affinity of these supports for insulin was determined by the establishment of adsorption isotherms and by high-performance affinity chromatography, to evidence the relationships between structural characteristics of the supports and their separation properties. The study of interactions between these supports and insulin allowed us to show the importance of the coupling method on the performances of supports in affinity chromatography.     

Purification of carbonic anhydrase isoenzymes by high-performance affinity chromatography and hydrophobic interaction chromatography

Isoenzymes of carbonic anhydrase were purified by a combination of affinity chromatography and hydrophobic interaction chromatography. Immobilization of sulfonamides on an epoxy-activated support provided a stationary phase for affinity chromatography which was stable to hydrolysis by carbonic anhydrase. A first purification step allowed the isolation of enzymes directly from homogenates of human erythrocytes and rat stomach. Without any further preparation, except the addition of ammonium sulfate to the eluate from affinity chromatography, the isoenzymes could be separated by hydrophobic interaction chromatography with very high recovery of protein and retention of enzymatic activity.     

Purification of catechol siderophores by boronate affinity chromatography: Identification of chrysobactin from Erwinia carotovora subsp. carotovora

Catechols are co-planar cis-diols known to form stable, isolable complexes with borate under weakly basic conditions. We exploited this chemistry and developed a boronate affinity chromatography for isolating catechol siderophores. The method was applied to the isolation of chrysobactin, enterobactin, and an unknown catechol siderophore produce by Erwinia carotovora subsp. carotovora W3C105. Yields of chrysobactin and enterobactin purified by boronate affinity chromatography were at least two-fold greater than those achieved through alternate methods. The unknown catechol produced by E. carotovora subsp. carotovora W3C105 was isolated by boronate affinity chromatography and shown to be identical to chrysobactin. Boronate affinity chromatography enabled separation of catechol from its rust-colored decomposition products, and simultaneous isolation of catechol and hydroxamate siderophores. Boronate affinity chromatography is a rapid and efficient method for purifying catechol siderophores from bacterial culture supernatants     

Quantitative affinity chromatography: increased versatility of the technique for studies of ligand binding

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.     

Partially polymerized erythrocyte actin obtained by affinity chromatography on DNAse sepharose. Comparison with rabbit skeletal muscle actin and role of calcium

A new procedure, consisting of affinity chromatography on DNAse sepharose, is worked out for the purification of human erythrocyte actin from an extract of acetone powder. Comparison of skeletal muscle and erythrocyte actin purified either by reversible polymerization or affinity chromatography on DNAse Sepharose led us to infer that the erythrocyte actin isolated by affinity chromatography was pure, devoid of spectrin, and was obtained in part under polymerized (di and tetrameric) forms. This partial polymerization is related to a loss of calcium bound to actin.     

Studies of lectin-carbohydrate interactions by quantitative affinity chromatography: systems with galactose and ovalbumin as saccharidic ligand

The potential of affinity chromatography for characterizing lectin-carbohydrate interactions is investigated. First, the effect of galactose on the chromatographic behavior of Ricinus communis phytohemagglutinin on Sepharose 4B is used to establish that quantitative affinity chromatography on polysaccharide matrices affords an unequivocal means of characterizing the interactions of lectins with monosaccharides in solution. Second, a method of characterizing lectin-glycoprotein interactions by affinity chromatography is illustrated in an experimental study with Sephadex G-50 as affinity matrix for examination of the interaction between concanavalin A and ovalbumin. Third, although no general solution to the problem of ligand multivalency in quantitative affinity chromatography has been found, an experimental protocol has been devised for the situation in which the partitioning solute (lectin) is univalent.     

Quantitative affinity chromatography.

The objective of this review is to summarize developments in the use of quantitative affinity chromatography to determine equilibrium constants for solute interactions of biological interest. Affinity chromatography is an extremely versatile method for characterizing interactions between dissimilar reactants because the biospecificity incorporated into the design of the affinity matrix ensures applicability of the method regardless of the relative sizes of the two reacting solutes. Adoption of different experimental strategies, such as column chromatography, simple partition equilibrium experiments, solid-phase immunoassay, and biosensor technology, has led to a situation whereby affinity chromatography affords a means of characterizing interactions governed by an extremely broad range of binding affinities--relatively weak interactions (binding constants below 10(3) M(-1)) through to interactions with binding constants in excess of 10(9) M(-1). In addition to its important role in solute separation and purification, affinity chromatography thus also possesses considerable potential for investigating the functional roles of the reactants thereby purified.     

Restoration of guanine nucleotide- and fluoride-stimulated activity to an adenylate cyclase-deficient cell line with affinity-purified guanine nucleotide regulatory protein.

A guanine nucleotide-binding protein purified from turkey erythrocytes by affinity chromatography confers both F-- and guanine nucleotide-stimulation of adenylate cyclase to membranes from CYC- cells, a mutant cell line deficient in these responses. Interaction of turkey erythrocyte membranes with beta-adrenergic agonists before affinity chromatography, which is essential for binding of the guanine nucleotide regulatory protein to the affinity matrix, was also required for recovery of F--stimulation restoring activity in the affinity eluate.     

免疫亲和层析法纯化苦瓜几丁酶

用扁豆几丁酶免疫家兔,获得抗扁豆几丁酶的抗体,将此抗体与Ｓｅｐｈａｒｏｓｅ ４Ｂ偶联,制备免疫亲和吸附剂,用以纯化苦瓜几丁酶．苦瓜叶片的粗提液经过免疫亲和吸附柱后,可获得电泳纯的几丁酶,其分子量为３５ ｋＤ,与用几丁质凝胶为亲和吸附剂的纯化结果一致．表明利用植物几丁酶在结构上的保守性,用免疫亲和法可纯化不同植物的同类几丁酶．与几丁质凝胶亲和柱相比,免疫亲和法纯化植物几丁酶具有快速、亲和柱可重复使用等的优点．利用免疫亲和层析获得的纯化样品,研究了苦瓜几丁酶对真菌的抑制试验,研究结果表明,苦瓜几丁酶能分解棉花枯萎病菌的菌丝体细胞壁制备物,并对其孢子芽管的伸长有一定抑制作用．     

Use of weak monoclonal antibodies for affinity chromatography

When using weakly interacting ligands in affinity chromatography, it is possible to take advantage of a true chromatographic process in the separation, as compared with traditional affinity chromatography which is rather an on/off process. In this work, weak monoclonal antibodies were immobilized on a silica and a perfusion-type support (POROS AL) and used for high-performance liquid affinity chromatography (HPLAC). Similar carbohydrate antigens were separated under isocratic conditions according to their weak interaction with the immobilized monoclonal antibody. The affinity of the antibodies was adjusted with temperature and pH to modify the separation. The productivity of the chromatographic system was increased with the immobilized perfusion support but at the expense of loss of plate numbers. This study clearly demonstrates the potential of weak affinity biological interactions as a basis for chromatographic separation.     

Dye-ligand affinity adsorbents for enzyme purification

Affinity chromatography is widely employed in laboratory and large-scale for the purification of biotherapeutics and diagnostics. Some of the most widely used ligands in affinity chromatography have been several reactive chlorotriazine dyes. In particular, immobilized anthraquinone dyes have found a plethora of applications in affinity chromatography because they are inexpensive, are resistant to chemical and biological degradation, are sterilizable and cleanable in situ, and are readily immobilized to generate affinity absorbents which display high binding capacity for a broad spectrum of proteins. This article provides detailed protocols on the preparation of a dye-ligand affinity adsorbent. Also, detailed protocols for effective application of these media, emphasizing binding and elution conditions are presented.     

用壳聚糖作为配基载体亲和层析提纯淀粉糖化酶的研究

以自制的壳聚糖作配基载体,植物血球凝集素(PHA)作配基,通过戊二醛交联研制成一种用于淀粉糖化酶提纯的新型亲和吸附剂。对淀粉糖化酶的亲和层析研究表明:提纯倍数为1.8,酶活性收率达80％,纯度经聚丙烯酰胺凝胶等电聚焦电泳(IEF-PAGE)鉴定为一条带,没有非特异性吸附作用。具有简单、安全、快速和高收率等优点。