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。     

Optimized operating parameters for the displacement separation of biomolecules in immobilized metal ion affinity chromatography

The ideal immobilized metal ion affinity chromatography (IMAC) model was employed to investigate the effect of operating parameters change on the displacement separation of biomolecules. By combining a lower initial mobile phase modifier (MPM) concentration and a higher final MPM concentration, the displacement chromatographic separation produced both higher concentration of feeds and better throughput in IMAC displacement separating systems.     

Genomic data for alternate production strategies. I. Identification of major contaminating species for Cobalt(+2) immobilized metal affinity chromatography

Recent advances in technology have allowed for the identification of complex protein mixtures in a rapid fashion. This report highlights the use of 2D gel electrophoresis, mass spectrometry, and database analysis to determine contaminating species of the Escherichia coli genome that are present during immobilized metal affinity chromatography (IMAC), highlighting Co(2+) as the affinity ligand. Four proteins (triosephosphate isomerase, alpha galactosidase, Hsp90, and glucosamine 6-phosphate synthase) constitute the majority of E. coli proteins that bind and potentially may coelute during chromatography. Results are discussed within the context of changes that when implemented could lead to an increase in IMAC efficiency, not by altering column conditions, but rather by changing the nature of the nuisance proteins that principally reduce column capacity and extend processing times. Such a study illustrates the use of proteome data to aid in bioprocess design.     

Phosphoproteomic analysis using immobilized metal ion affinity chromatography on the basis of cellulose powder

Detailed characterization of phosphoproteins as well as other post-translationally modified proteins such as glycoproteins, is required to fully understand protein function and regulatory events in cells and organisms. Therefore, an experimental strategy for the isolation of phosphoproteins using a new immobilized metal ion affinity chromatograph (IMAC) material on the basis of cellulose has been developed and characterized. Different approaches have been used to test the material. Recovery rates were determined by 32P labelling of a myelin basic protein fragment and by reversed-phase high-performance liquid chromatography-electrospray ionization mass spectrometry using a tryptic digest of the model protein bovine beta-casein. Selectivity was demonstrated by enrichment and separation of phosphopeptides from different samples, such as from a digest of horse myoglobin as well as from a digest of in vitro phosphorylated extracellular signal regulates kinase 2 (ERK2) mixed with synthetic phosphopeptides, phosphorylated on different amino acid residues. Furthermore, simplification and optimization of sample pretreatment was achieved by combining the separating (IMAC) and desalting (C18) step during preparative high performance liquid chromatography. The comparison between our material and a commercially available IMAC system (POROS 20 MC; Perspective BioSystems) emphasizes the competitiveness of the cellulose. Confirmed by the obtained data, the cellulose material performed as well as the commercially available sorbent, however with the advantage, that it can be produced rather easily and at very low cost.     

Purification of a recombinant protein produced in a baculovirus expression system by immobilized metal affinity chromatography

Over the past 10 years, the baculovirus-insect cell system has become a powerful and versatile tool for the expression of a variety of heterologous proteins. In order to simplify separation of a cloned protein from the baculovirus-insect expression system, we have cloned a gene encoding for the protein of interest, a structural protein (VP2) of a strain (E/DEL) of infectious bursal disease virus (IBDV), with a metal ion binding site (His)(5) at its C-terminus. This chimeric protein (VP2H) has been expressed and one-step affinity purified with immobilized metal ions (Ni(+2)). With antigen capture-enzyme-linked immunosorbent assay (AC-ELISA), we determined that the conformation of this chimeric protein was no different from the recombinant wild-type VP2 protein. However, the two proteins (VP2 and VP2H) can be distinguished and resolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and detected immunologically following Western blotting. (c) 1994 John Wiley & Sons, Inc.     

Review: Multipoint binding and heterogeneity in immobilized metal affinity chromatography

Studies carried out using engineered proteins clearly demonstrate that adsorption to derivatized surfaces involves multiple interactions between functional groups on the protein and complementary sites distributed on the surface. The fact that adsorption involves multipoint interactions has important implications for the design of separations processes and for the interpretation of heterogeneity in biological recognition phenomena. Increasing the density of surface metal sites (immobilized copper ions) is found to be functionally equivalent to increasing the number of metal-coordinating groups on the protein (histidines and deporotonated amines), m in that both processes increase the likelihood of simultaneous interactions between the protein and the surface. A consequence of multiple-site interactions is a significant in crease in protein binding affinity that depends on the arrangement of surface sites. A protein will show the highest affinity for arrangements of surface sites which best match its own pattern of functioal groups and will show lower affinity for less optimal arrangements, resulting in binding that is inherently heterogeneous. We have found that reversible protein adsorption in immobilized metal affinity chromatography (IMAC) is described by the Temikin model, which characterizes binding heterogeneity by a uniform distribution of binding energies over the population of surface binding sites. (c) 1995 John Wiley & Sons, Inc.     

A modified IMAC method for the capture of target protein from mammalian cell culture harvest containing metal chelating species

Although immobilized metal affinity chromatography (IMAC) offers high capacity and protein selectivity it is not typically used commercially for the capture of native proteins from mammalian cell culture harvest. This is due mainly to the potential for low target recovery due to the presence of strong metal ion chelating species in the harvest that compete for the metal immobilized on the resin. To address this issue a buffer exchange step, such as tangential flow filtration (TFF), is added after harvest clarification and prior to IMAC to remove the interfering harvest components. The addition of a TFF step adds process time and cost and reduces target protein recovery. The elimination of the TFF might make IMAC competitive with other orthogonal methods of protein capture. In this study, we developed a modified IMAC method to allow the direct loading of clarified mammalian harvest without prior buffer exchange (direct IMAC). Although the target enzyme recovery was lower than that from standard IMAC the elimination of the buffer exchange step resulted in a 19% increase in overall enzyme recovery. The target enzyme capacity in direct IMAC was higher, in our experience, than the capacity of hydrophobic interaction (HIC) and ion-exchange (IEX) for protein capture. An economic evaluation of using direct IMAC as a capture step in manufacturing is also discussed.     

Optimization of phosphopeptide elution conditions in immobilized Fe(III) affinity chromatography

While immobilized metal affinity chromatography (IMAC) has been widely used for affinity purification of phosphopeptides, the technique suffers from insufficient specificity. Therefore, there is an urgent need for IMAC optimization to yield the selectivity and sensitivity that is required for more challenging analyses. Recently, 2,5-dihydroxybenzoic acid (DHB) and phosphoric acid mixture has been reported as an efficient IMAC eluant. The disadvantage of DHB is that is not suitable for electrospray ionization-mass spectrometry. While further developing the IMAC elution protocol to overcome this problem, we noticed that DHB is not necessary and found a novel combination of phosphoric acid and acetonitrile to be more efficient. The purification efficacy of the novel protocol is superior to all previously described methods, while still being compatible with the most commonly used mass-spectrometric techniques in phosphoproteomics.     

Purification of penicillin G acylase using immobilized metal affinity membranes

The immobilized metal affinity membrane (IMAM) with modified regeneration cellulose was employed for purification of penicillin G acylase (PGA). For studying PGA adsorption capacity on the IMAM, factors such as chelator surface density, chelating metal, loading temperature, pH, NaCl concentration and elution solutions were investigated. The optimal loading conditions were found at 4 degrees C, 0.5 M NaCl, 32.04 micromol Cu(2+) per disk with 10 mM sodium phosphate buffer, pH 8.5, whereas elution conditions were: 1 M NH(4)Cl with 10 mM sodium phosphate buffer, pH 6.8. By applying these chromatographic conditions to the flow experiments in a cartridge, a 9.11-fold purification in specific activity with 90.25% recovery for PGA purification was obtained. Meanwhile, more than eight-times reusability of the membrane was achieved with the EDTA regeneration solutions.     

Rational design and synthesis of affinity matrices based on proteases immobilized onto cellulose membranes

Discovery of new protease inhibitors may result in potential therapeutic agents or useful biotechnological tools. Obtainment of these molecules from natural sources requires simple, economic, and highly efficient purification protocols. The aim of this work was the obtainment of affinity matrices by the covalent immobilization of dipeptidyl peptidase IV (DPP-IV) and papain onto cellulose membranes, previously activated with formyl (FCM) or glyoxyl groups (GCM). GCM showed the highest activation grade (10.2?µmol aldehyde/cm²). We implemented our strategy for the rational design of immobilized derivatives (RDID) to optimize the immobilization. pH 9.0 was the optimum for the immobilization through the terminal α-NH₂, configuration predicted as catalytically competent. However, our data suggest that protein immobilization may occur via clusters of few reactive groups. DPP-IV?GCM showed the highest maximal immobilized protein load (2.1?µg/cm²), immobilization percentage (91%), and probability of multipoint covalent attachment. The four enzyme-support systems were able to bind at least 80% of the reversible competitive inhibitors bacitracin/cystatin, compared with the available active sites in the immobilized derivatives. Our results show the potentialities of the synthesized matrices for affinity purification of protease inhibitors and confirm the robustness of the RDID strategy to optimize protein immobilization processes with further practical applications.     

生物质谱结合IMAC亲和提取和磷酸酶水解分析蛋白质磷酸化修饰

蛋白质磷酸化是生物体内非常重要的翻译后修饰方式 ,对磷酸化蛋白质的分析及磷酸化位点的确定有助于理解与其相关的生物功能。基质辅助激光解吸 /电离飞行时间质谱和电喷雾 四极杆 飞行时间质谱这两种生物质谱仪在蛋白质鉴定和翻译后修饰分析中发挥着重要作用。固相金属亲和色谱可选择性亲和提取肽混合物中的磷酸肽 ,结合磷酸酶水解实验和基质辅助激光解吸 /电离飞行时间质谱分析可确定肽混合物中的磷酸肽 ,最后用电喷雾 四极杆 飞行时间串联质谱分析磷酸肽的序列 ,结合数据库检索确定磷酸化位点。     

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     

Fe3+ immobilized metal affinity chromatography with silica monolithic capillary column for phosphoproteome analysis

Immobilized metal affinity chromatography (IMAC) is a commonly used technique for phosphoproteome analysis due to its high affinity for adsorption of phosphopeptides. Miniaturization of IMAC column is essential for the analysis of a small amount of sample. Nanoscale IMAC column was prepared by chemical modification of silica monolith with iminodiacetic acid (IDA) followed by the immobilization of Fe3+ ion inside the capillary. It was demonstrated that Fe3+-IDA silica monolithic IMAC capillary column could specifically capture the phosphopeptides from tryptic digest of alpha-casein with analysis by MALDI-TOF MS. The silica monolithic IMAC capillary column was manually coupled with nanoflow RPLC/nanospray ESI mass spectrometer (muRPLC-nanoESI MS) for phosphoproteome analysis. The system was validated by analysis of standard phosphoproteins and then it was applied to the analysis of protein phosphorylation in mouse liver lysate. Besides MS/MS spectra, MS/MS/MS spectra were also collected for neutral loss peak. After database search and manual validation with conservative criteria, 29 singly phosphorylated peptides were identified by analyzing a tryptic digest of only 12 mug mouse liver lysate. The results demonstrated that the silica monolithic IMAC capillary column coupled with muRPLC-nanoESI MS was very suitable for the phosphoproteome analysis of minute sample.     

Activity of immobilized papain dehydrated by n-propanol in low-water media

A propanol-rinsed enzyme preparations (PREP) of papain showed an activity of 59 nmol min(-1) (mg powder)(-1) in tert-butanol at the optimal water activity of 0.2. The immobilized papain was stable in aqueous media for 3 d at 4 degrees C. Solid-state buffers (bases and their HCl salts) suspended in the organic medium decreased the initial rate in all cases tested. The operational stability during Z-Gly-Phe-NH2 synthesis was improved when solid cysteine was added, doubling the yield after 24 h.     

双醛淀粉柔性固定木瓜蛋白酶研究

提出“柔性固定化酶”的模型,即：用一亲水、柔性高分子链接枝于载体表面制得柔性固定化载体,再用其以共价键合的方式进行酶的柔性固定化。其特点是：柔性固定可改善因直接固定化及手臂固定化使酶失活的缺陷,并提高固定化酶的自由度;如选用粒径单分散微球可改善固定化反应及固定化酶催化反应的均一性。以双醛淀粉(DAS)为柔性链对羧基化聚苯乙烯载体进行柔性化修饰后,固定木瓜蛋白酶,其活力回收率可达50％．相当于用戊二醛进行手臂固定化的活力回收率的2倍。     

A novel method to quantify sphingosine 1-phosphate by immobilized metal affinity chromatography (IMAC)

Sphingosine 1-phosphate (S1P), a lysophospholipid mediator that signals through G protein-coupled receptors, regulates a wide plethora of biological responses such as angiogenesis and immune cell trafficking. Detection and quantification of S1P in biological samples is challenging due to its unique physicochemical nature and occurrence in trace quantities. In this report, we describe a new method to selectively enrich S1P and dihydro-S1P from biological samples by the Fe(3+) gel immobilized metal affinity chromatography (IMAC). The eluted S1P from IMAC was dephosphorylated, derivatized with o-phthalaldehyde (OPA), and detected by high-performance liquid chromatography (HPLC) coupled to a fluorescence detector. IMAC purification of S1P was linear for a wide range of S1P concentration. Using this assay, secretion of endogenous S1P from endothelial cells, fibroblasts and colon cancer cells was demonstrated. We also show that dihydro-S1P was the major sphingoid base phosphate secreted from HUVEC over expressed with Sphk1 cDNA. Pharmcological antagonists of ABC transporters, glyburide and MK-571 attenuated endogenous S1P release. This assay was also used to demonstrate that plasma S1P levels were not altered in mice deficient for ABC transporters, Abca1, Abca7 and Abcc1/Mrp1. IMAC-based affinity-enrichment coupled with a HPLC-based separation and detection system is a rapid and sensitive method to accurately quantify S1P.     

Proteomic survey of copper-binding proteins in Arabidopsis roots by immobilized metal affinity chromatography and mass spectrometry

To plants, copper is vitally essential at low concentrations but extremely toxic at elevated concentrations. Plants have evolved a suite of mechanisms that modulate the uptake, distribution, and utilization of copper ions. These mechanisms require copper-interacting proteins for transporting, chelating, and sequestrating copper ions. In this study, we have systematically screened for copper-interacting proteins in Arabidopsis roots via copper-immobilized metal affinity chromatography (Cu-IMAC). We also compared Arabidopsis root metalloproteomes with affinity to Cu-IMAC and Zn-IMAC. From the identities of 38 protein spots with affinity to Cu-IMAC, 35 unique proteins were identified. Functional classification of these proteins includes redox/hydrolytic reactions, amino acid metabolism, glutathione metabolism, phosphorylation, translation machinery, membrane-associated proteins, and vegetative storage proteins. Potential copper-interacting motifs were predicted and scored. Six candidate motifs, H-(X)5 -H, H-(X)7 -H, H-(X)12 -H, H-(X)6 -M, M-(X)7 -H, and H-(X)3 -C, are present in Cu-IMAC-isolated proteins with higher frequency than in the whole Arabidopsis proteome.     

Purification of human liver glycoprotein sialyltransferase by affinity chromatography

A simple procedure has been developed for purifying solubilized human liver glycoprotein sialyltransferase (EC 2.4.99.1) 16 000-fold in 4–5% yield. The procedure involves two centrifugation steps, affinity chromatography of the ultrasupernatant fluid on cytidine diphosphate-hexanolamine-agarose followed by gel filtration on Sephadex G-150. Sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) indicated that the purified sialyltransferase preparation contains approximately equivalent amounts of three protein bands (with apparent molecular weights of 61 000, 63 000 and 70 000) and is highly purified if not homogeneous.     

Immobilized metal affinity chromatography in the presence of arginine

Arginine hydrochloride (ArgHCl) is a versatile solvent additive, as it suppresses protein aggregation. ArgHCl has been used for protein refolding and to solubilize proteins from loose inclusion bodies. Immobilized metal affinity chromatography (IMAC) is one of the most commonly used technologies for purification of recombinant proteins. Here we have evaluated compatibility of ArgHCl with IMAC purification for his-tag proteins. ArgHCl clearly interfered with protein binding to Ni-columns. Nevertheless, such interference was greatly reduced at ArgHCl concentration below 200 mM, demonstrating that IMAC purification can be done even in the presence of ArgHCl.