重组人Fab金属螯合金层析法纯化条件的研究

在重组人Ｆａｂ表达载体的羧基端插入六个组氨酸,使其对金属螯合层析介质产生特异性吸附,可用金属螯合亲和层析法进行分离纯化。采用自制金属螯合层析介质,以ＰＨ和咪唑两种脱方法,对ｒｈＦａｂ段的纯效果进行了探讨。     

金属螯合亲和层析法纯化抗乙肝核心抗原单克隆抗体

采用金属螯合亲和层析法,纯化了小鼠腹水来源的抗乙肝核心抗原单克隆抗体,对上样缓冲液的pH和离子强度、洗脱液种类和洗脱方式进行优化。结果表明,采用降低pH分步洗脱时,最佳上样缓冲液为pH8.0,20mmol/LPB+0.5mol/LNaCl,抗体在pH5.0被洗脱下来,抗体回收率80%,纯度85%。采用咪唑浓度梯度洗脱时,最佳的上样缓冲液为pH8.0,20mmol/LPB+5mmol/L咪唑,抗体纯度大于95%,回收率65%;在上样缓冲液中不添加NaCl而添加少量的咪唑,更有利于抗体分离。以上洗脱方式都能较好地保持mAb的生物学活性,为该抗体的应用提供了必要的实验基础。     

金属螯合亲和层析纯化金属硫蛋白

将二价铜离子螯合在Chelating Sepharose Fast Flow凝胶上制成亲和层析柱,锌诱导兔肝和镉诱导小鼠肝经匀浆、乙醇处理后上柱,用pH4.0的醋酸盐缓冲液平衡,再用pH5.2不同浓度的醋酸盐缓冲液分别洗脱,可得两个金属硫蛋白(MT)洗脱峰,经确定先后为MT-2和MT-1.分离方法比传统的凝胶过滤-离子交换法简单、省时,适于实验室规模分离纯化.     

以壳聚糖为载体金属螯合亲和层析法纯化猪红细胞Cu·Zn—SOD

从虾壳制备所得壳聚糖为基质合成金属螫合亲和吸附剂,用于纯化猪血铜锌超氧化物歧化酶(Cu·Zn-SOD),得到比活为4756U·mg-1的酶蛋白,收率为67%,纯化倍数为6.1.聚丙烯酰胺凝胶电泳及活性染色定位表明,经螯合层析纯化后的酶纯度基本均一.     

坛紫菜中别藻蓝蛋白的纯化及洗脱组分光谱研究

以福建省平潭县坛紫菜(Porphyra haitanensis)为材料,采用硫酸铵分级分离和柱层析法纯化别藻蓝蛋白(APC),并对纯化的条件进行了详细的探讨。研究结果表明:采用30%~35%饱和硫酸铵沉淀、Tris-HC l(pH=8.0)作为洗脱缓冲液、DEAE-Sephadex-A-50作为层析介质,所获得的APC的纯度和回收率分别为3.50和70.2%;SDS-PAGE表明APC有α和β两个亚基,分子量分别为18.9 kD和17.7 kD。因此该方法对于从坛紫菜中快速纯化APC是适合的。采用光谱分析研究柱洗脱组分,结果表明:坛紫菜中含有分子结构为(αβ)6γ的“双峰型”R-PE,含有结构为(αβ)3的R-PC,含有结构为(αβ)3的APC-Ⅱ。     

固定化金属螯合亲和膜纯化重组抗菌肽研究

用自行制备的固定化金属螯合亲和膜对N端含六个组氨酸标记的重组抗菌肽进行了分离纯化,并较好地解决了金属离子泄露问题.实验表明,固定化金属螯合亲和膜性能优于传统琼脂糖凝胶介质,完全适用于分离纯化含有多组氨酸标记的重组蛋白质.     

用膨胀床金属亲和层析从淡菜匀浆液中分离纯化纤维素酶

研究了一种新的膨胀床金属亲和层析技术,即将金属亲和层析结合膨胀床层析,直接从淡菜(Blue mussel)匀浆液中纯化纤维素酶。研究了金属亲和配基种类、pH、离子强度及流速对酶吸附和解吸的影响,确定了酶洗脱条件和介质再生条件。一步可纯化纤维素酶194倍,酶收率达82%。本方法不需要预先去除细胞碎片,而且处理速率比传统层析技术高3～4倍。     

金属螯合亲和层析分离蛋白质的研究

金属螯合亲和层析是近20年发展起来的一项新型分离技术。它以配基简单、吸附量大、分离条件温和、通用性强等特点,逐渐成为分离纯化蛋白质等生物工程产品最有效的技术之一。本文从单组分蛋白质入手,考查了pH值、铵离子浓度、不同铵盐等对蛋白质洗脱的影响,并进行了分析。还对不同的金属螯合柱和不同性质蛋白质的洗脱性能进行了研究,比较了不同金属离子与蛋白质亲和力的区别,为实际体系的分离研究打下了基础。     

铜离子螯合亲和层析纯化人铜锌超氧化物歧化酶

用铜离子螯合亲和层析对人红细胞铜锌超氧化物歧化酶进行了纯化．3次实验的结果表明,此项层析具有重复使用率高和蛋白结合量大的显著优点．提纯的人铜锌超氧化物歧化酶的比活性为3037U每毫克蛋白,并经活性染色和SDS聚丙烯酰胺凝胶电泳证实其纯度均一．纯化中,探索了用紫外260nm与280nm的A比值判断酶纯度的简便方法．     

酶标免疫分析法鉴定蛇毒

蛇伤的治疗与蛇伤种类的快速诊断密切相关。我们用亲和层析法纯化了三种蛇毒的抗体Fab,它们之间无免疫交叉反应。然后用高碘酸钠法将辣根过氧化物酶标记到抗体Fab上。我们建立的三夹心式酶标免疫测定蛇毒的方法,可在90分钟内检测到5毫微克以下的蛇毒,人体血清对该测定无干扰现象,临床检测效果良好。     

Effect of metal ions on sucrose synthase from rice grains--a study on enzyme inhibition and enzyme topography

The inhibition of the plant glycosyltransferase sucrose synthasefrom rice grains by free metal ions was studied. Decreasingsucrose synthase activities in the order of metal ions (Cu²⁺>> Zn²⁺ Ni²⁺ > Fe²⁺; 15·4% residual activitywith 30 µM Cu²⁺) as well as inhibition by diethyl pyrocarbonate(27% residual activity at pH 7·2 and 43 µM diethylpyrocarbonate) provided evidence that histidyl residues areimportant for sucrose synthase activity. Chelated metal ions,due to the geometric restriction of the reagent, gave a lesspronounced inhibitory effect (11·7% residual activitywith 100 µM Cu²⁺), but suggested that surface-accessiblehistidine residues are probably involved. Inhibition of sucrosesynthase could always be prevented by metal ion scavengers [ethylenediaminetetra-aceticacid (EDTA), dithiothreitol (DTT), mercaptoethanol, reducedglutathione, imidazole and histidine]. Sucrose synthase inhibitedby free and chelated Cu²⁺, respectively, could be partly (60%)reactivated by EDTA. These results led to a topographical analysisof histidines on the surface of the homotetrameric protein byimmobilized metal ion chromatography (IMAC). From the orderby which sucrose synthase was bound to immobilized chelatedmetal ions in the presence of 1 mM imidazole (Cu²⁺ > Ni²⁺> Zn²⁺ = Co²⁺), it could be concluded that the enzyme hasat least 5–7 surface-accessible histidines. Sucrose synthasecould not be eluted from a Cu²⁺ column by an increasing imidazolegradient. These results are of particular interest for the furtherpurification of sucrose synthase(s), as well as for the evaluationof cloning and expression strategies using poly-hLstidine tails. glycosyltransferase IMAC nucleotide sugars sucrose synthase     

Evaluation of immobilized metal affinity chromatography for purification of penicillin acylase

The aim of this work was to test immobilized metal affinity chromatography (IMAC) for the purification of penicillin acylase. After evaluation of different metals, Cu²⁺ was selected. Different samples were tested: pure penicillin acylase, industrial clarified feedstock and crude extract. After comparing two eluents, NH₄Cl and imidazole, it appeared that although both gave good results for recovery and activity, NH₄Cl was a more selective eluent with a higher fold purification than imidazole (4.64 versus 2.04). Moreover, we shown that a multistep gradient of NH₄Cl, greatly increased the degree of purification (12.36) compared with the one-step process as control (4.64). In addition, good recovery was obtained (97–100%).     

Protein selectivity in immobilized metal affinity chromatography based on the surface accessibility of aspartic and glutamic acid residues

The interaction of different species variants of cytochrome c and myoglobin, as well as hen egg white lysozyme, with the hard Lewis metal ions Al³⁺, Ca²⁺, Fe³⁺, and Yb³⁺ and the borderline metal ion Cu²⁺, immobilized to iminodiacetic acid (IDA)-Sepharose CL-4B, has been investigated over the rangepH 5.5–8.0. With appropriately chosen buffer and metal ion conditions, these proteins can be bound to the immobilized M ⁿ +-IDA adsorbents via negatively charged amino acid residues accessible on the protein surface. For example, tuna heart cytochrome c, which lacks surface-accessible histidine residues, readily bound to the Fe³⁺-IDA adsorbent, while the other proteins also showed affinity toward immobilized Fe³⁺-IDA adsorbents when buffers containing 30 mM of imidazole were used. These studies document that protein selectivity can be achieved with hard-metalion immobilized metal ion affinity chromatography (IMAC) systems through the interaction of surfaceexposed aspartic and glutamic acid residues on the protein with the immobilized M ⁿ +-IDA complex. These investigations have also documented that the so-called soft or borderline immobilized metal ions such as the Cu²⁺-IDA adsorbent can also interact with surface-accessible aspartic and glutamic acid residues in a protein-dependent manner. A relationship is evident between the number and extent of clustering of the surfaceaccessible aspartic and glutamic acid residues and protein selectivity with these IMAC systems. The use of elution buffers which contain organic compound modifiers which replicate the carboxyl group moieties of these amino acids on the surface of proteins is also described.Abbreviations IDA iminodiacetic acid - IDA-Mⁿ⁺ iminodiacetic acid chelated to metal ion - IMAC immobilized metal affinity chromatography - DHCC dog heart cytochrome c - HHCC horse heart cytochrome c, THCC, tuna heart cytochrome c - HMYO horse skeletal muscle myoglobin - SMYO sheep skeletal muscle myoglobin - HEWL hen egg white lysozyme     

Separation of hexahistidine fusion proteins with immobilized metal ion affinity chromatographic (IMAC) sorbents derived from MN+‐tacn and its derivatives

The capabilities of a new class of immobilized (im) metal ion chelate complexes (IMCCs), derived from 1,4,7‐triazacyclononane (tacn), bis(1,4,7‐triazacyclononyl) ethane (dtne) and bis(1,4,7‐triazacyclononyl)propane (dtnp) complexed with the borderline metal ions Cu²⁺, Ni²⁺, Zn²⁺, Mn²⁺, Co²⁺, and Cr³⁺, for the purification of proteins have been investigated. In particular, the binding behavior of a model protein, the C‐terminal hexahistidine tagged recombinant fusion protein Schistosoma japonicum glutathione S‐transferase‐Saccharomyces cerevisiae mitochondrial ATP synthase δ‐subunit (GST‐δATPase‐His₆), with these new immobilized metal ion affinity chromatographic (IMAC) sorbents was compared to the properties of a conventional sorbent, derived from immobilized Ni(II)‐nitrilotriacetic acid (im‐Ni²⁺‐NTA). Investigations using the recombinant GST‐δATPase‐His₆ and recombinant S. japonicum glutathione S‐transferase (GST) lacking a hexahistidine tag have confirmed that the C‐terminal tag hexahistidine residues were required for the binding process to occur with these IMAC systems. The results also confirm that recombinant fusion proteins such as GST‐δATPase‐His₆ can be isolated in high purity with these IMAC systems. Moreover, these new macrocyclic systems manifest different selectivity features as a function of pH or ionic strength when compared to the conventional, unconstrained iminodiacetic acid (IDA) or NTA chelating ligands, complexed with borderline metal ions such as Cu²⁺ or Ni²⁺, as IMAC systems. Biotechnol. Bioeng. 2009;103: 747–756. © 2009 Wiley Periodicals, Inc.     

Polymer versus monomer as displacer in immobilized metal affinity chromatography

Successful immobilized metal affinity chromatography (IMAC) of proteins on Cu²⁺-iminodiacetic acid Sepharose has been carried out in a displacement mode using a synthetic copolymer of vinyl imidazole and vinyl caprolactam [poly(VI-VCL)] as a displacer. Vinyl caprolactam renders the co-polymer with the thermosensitivity, e.g., property of the co-polymer to precipitate nearly quantitatively from aqueous solution on increase of the temperature to 48°C. A thermostable lactate dehydrogenase from the thermophilic bacterium Bacillus stearothermophilus modified with a (His)₆-tag [(His)₆-LDH] has been purified using an IMAC column. For the first time it was clearly demonstrated that a polymeric displacer [poly(VI-VCL)] was more efficient compared to a monomeric displacer (imidazole) of the same chemical nature, probably due to the multipoint interaction of imidazole groups within the same macromolecule with one Cu²⁺ ion. Complete elution of bound (His)₆-LDH has been achieved at 3.7 mM concentration of imidazole units of the co-polymer (5 mg/ml), while this concentration of free imidazole was sufficient to elute only weakly bound proteins. Complete elution of (His)₆-LDH by the free imidazole was achieved only at concentrations as high as 160 mM. Thus, it was clearly demonstrated, that the efficiency of low-molecular-mass displacer could be improved significantly by converting it into a polymeric displacer having interacting groups of the same chemical nature.     

High-performance analytical applications of immobilized metal ion affinity chromatography

The separation of three sets of standard protein mixtures on a high-performance immobilized metal ion affinity chromatography (HP-IMAC) column by elution with linear gradients of imidazole is described. The affinity of the test proteins for the immobilized metal ions follows the order Cu2+ greater than Ni2+ greater than Zn2+. The iminodiacetic acid-Cu2+ column gives the best resolution of all three protein mixtures and is the only immobilized metal ion column that can be used for elution of absorbed proteins with a decreasing pH gradient. An application of HP-IMAC for the separation of monoclonal IgG from mouse ascites fluid is also outlined. This versatile separation method is thus suitable for both analytical and preparative separations of proteins and peptides resulting in high recoveries and good reproducibility. The leakage of immobilized metal ions from the TSK gel chelate-5PW is apparent if the column is eluted by buffers containing low concentrations of (i) glycine or (ii) primary amines at round neutral pH. Considerable amounts of immobilized Zn2+ and Ni2+ ions also leak from the column by washing with buffers of pH 4.5 or lower. However, all three immobilized metal ions are stable toward exposure to low concentrations of imidazole (up to 50 mM) in phosphate buffers between pH 6.5 and 8.0. Adsorbed proteins could thus be eluted conveniently by using linear gradients of imidazole to give reproducible results. Moreover, this elution procedure made it possible to use the IMAC columns for repeated runs without the need for regeneration and recharging of the columns with fresh metal ions after each use.     

Use of Streamline chelating for capture and purification of poly-His-tagged recombinant proteins

Expression of recombinant proteins with poly-histidine tags enables their convenient capture and purification using immobilized metal affinity chromatography (IMAC). The 6×His-tagged protein binds to a chelating resin charged with metal ions such as Ni²⁺, Cu²⁺ or Zn²⁺, and can therefore be separated from proteins which have lower, or no, affinity for the resin. Two recombinant proteins, a malaria transmission-blocking vaccine candidate secreted extracellularly by S. cerevisiae and a modified diphtheria toxin produced intracellularly by E. coli, were expressed with 6×His tags and could therefore be purified using IMAC. In an effort to further simplify the initial capture of these proteins, an expanded bed adsorption technique using a chelating resin (Streamline Chelating) was introduced. It was possible to capture the intracellular diphtheria protein from E. coli directly after cell lysis, without prior centrifugation or filtration. The extracellular malaria vaccine candidate was also directly captured from a high cell density yeast culture. Detailed information on the experimental work performed, and the capture processes developed, is provided.     

Reversible immobilization of invertase on Cu-chelated polyvinylimidazole-grafted iron oxide nanoparticles

Polyvinylimidazole (PVI)-grafted iron oxide nanoparticles (PVIgMNP) were prepared by grafting of telomere of PVI on the iron oxide nanoparticles. Different metal ions (Cu²⁺, Zn²⁺, Cr²⁺, Ni²⁺) ions were chelated on polyvinylimidazole-grafted iron oxide nanoparticles, and then the metal-chelated magnetic particles were used in the adsorption of invertase. The maximum invertase immobilization capacity of the PVIgMNP–Cu²⁺ beads was observed to be 142.856 mg/g (invertase/PVIgMNP) at pH 5.0. The values of the maximum reaction rate (V _max) and Michaelis–Menten constant (Km) were determined for the free and immobilized enzymes. The enzyme adsorption–desorption studies, pH effect on the adsorption efficiency, affinity of different metal ions, the kinetic parameters and storage stability of free and immobilized enzymes were evaluated.     

Resolution of three forms of superoxide dismutase by immobilised metal affinity chromatography

A new method for separation of three forms of superoxide dismutase (SOD) using immobilised metal affinity chromatography (IMAC) is reported. Fe-, Mn- and Cu/Zn-SODs were eluted sequentially from Cu²⁺-IMAC column with an increasing gradient of a counter ion (NH⁺₄) run in combination with an increasing pH gradient (6.8–7.8). The combined gradient elution method resulted in separation of SODs with high resolution, the three proteins being eluted in electrophoretically homogeneous forms. Similar preparation could not be achieved by either increasing gradient of a counter ion or decreasing pH gradients used separately. The described methodology has been successfully applied for separation of three SODs from a protozoan parasite, indicating that this combined gradient elution system for IMAC offers new possibilities for the high-resolution separation of proteins exhibiting only minor differences in their amino acid composition and structure.     

Anti-fibrillogenic and fibril destabilizing effects of metal ions on cystatin fibrils

Metals such as Cu²⁺, Fe³⁺, and Zn²⁺ are major contributors to the biology of a brain in stages of health, aging, and disease because of their unique effects on both protein structures (misfolding) and oxidative stress. The relationship between metal ions and neurodegenerative diseases is very complicated. Our study highlights how metal ions influence amyloid formation at low pH and on preformed amyloid fibrils. By using thioflavin T assay, ANS fluorescence, Congo red assay, circular dichroism, and microscopy to elucidate the effects of Cu²⁺, Fe³⁺, and Zn²⁺ on goat brain cystatin (GBC) aggregation at low pH. Results showed that Cu²⁺ and Fe³⁺ inhibit fibril formation of GBC by promoting amorphous aggregates. However, Zn²⁺ exclusively promotes fibril formation at low pH, leading to the formation of more ordered aggregates. Furthermore, the combined results of these complementary methods also suggested that Cu²⁺ and Fe³⁺ destabilize the β-sheet secondary structure of preformed amyloid fibrils of GBC.