Showing papers on "Affinity chromatography published in 2007"
TL;DR: The Strep-tag II, which is short, biologically inert, proteolytically stable and does not interfere with membrane translocation or protein folding, offers a versatile tool both for the rapid isolation of a functional gene product and for its detection or molecular interaction analysis.
Abstract: The Strep-tag II is an eight-residue minimal peptide sequence (Trp-Ser-His-Pro-Gln-Phe-Glu-Lys) that exhibits intrinsic affinity toward streptavidin and can be fused to recombinant proteins in various fashions. We describe a protocol that enables quick and mild purification of corresponding Strep-tag II fusion proteins--including their complexes with interacting partners--both from bacterial and eukaryotic cell lysates using affinity chromatography on a matrix carrying an engineered streptavidin (Strep-Tactin), which can be accomplished within 1 h. A high-affinity monoclonal antibody (StrepMAB-Immo) permits stable immobilization of Strep-tag II fusion proteins to solid surfaces, for example, for surface plasmon resonance analysis. Selective and sensitive detection on western blots is achieved with Strep-Tactin/enzyme conjugates or another monoclonal antibody (StrepMAB-Classic). Thus, the Strep-tag II, which is short, biologically inert, proteolytically stable and does not interfere with membrane translocation or protein folding, offers a versatile tool both for the rapid isolation of a functional gene product and for its detection or molecular interaction analysis.
597 citations
TL;DR: A minimized SPA derivative has been constructed and a domain originating from SPA has been improved to withstand the harsh environment employed in industrial purifications.
519 citations
TL;DR: 35 proteins, of which 24 are intact, were found to be associated with albumin, and they include both known high and low abundance proteins.
Abstract: The removal of albumin and other high abundance proteins is a routine first step in the analysis of serum and plasma proteomes. However, as albumin can bind proteins and peptides, there is a universal concern as to how the serum proteome is changed by the removal of albumin. To address this concern, the current study was designed to identify proteins and peptides removed from the serum during albumin depletion; to determine which of these are bound to albumin (rather than copurified) and whether the bound proteins are intact proteins or peptide fragments. Sequential, independent analyses including both anti-albumin antibody (anti-HSA) affinity chromatography and SEC were used to isolate albumin-bound proteins. RP-HPLC and 1-D SDS-PAGE were then used to further separate the proteins prior to identification by MS/MS. Finally, whole protein molecular weight (MW) MS measurements coupled with protein coverage obtained by MS were combined to assess whether the bound proteins were intact or peptide fragments. Combining the results from multiple approaches, 35 proteins, of which 24 are intact, were found to be associated with albumin, and they include both known high and low abundance proteins.
175 citations
TL;DR: Several cancer‐selective proteins that have been previously characterized as potential indicators of lung cancer in serum or plasma are identified, including haptoglobin, inter‐α‐trypsin inhibitor heavy chain 4, complement C3 precursor, and leucine‐rich α‐2‐glycoprotein.
Abstract: Glycoproteins in human serum play fundamental roles in many biological processes, and also have clinical value as biomarkers for disease progression and treatment. In this study, we isolated glycoproteins from the sera of three healthy individuals and three lung adenocarcinoma patients using multilectin affinity chromatography. The recovered glycoproteins were subjected to treatment with peptide-N-glycosidase F (PNGase F) and in-gel digestion by trypsin. Tryptic peptides were analyzed by nano-LC coupled to ESI-MS/MS and the MS/MS spectra were processed by Bioworks 3.2 and an in-house bioinformatics tool, ProtAn. Approximately 90% of the proteins identified contained more than one potential glycosylation site. Comparison of the serum glycoproteome of healthy and adenocarcinoma individuals revealed 38 cancer-selective proteins. Among them, 60% have previously been reported as low abundance proteins in human sera. We identified several cancer-selective proteins that have been previously characterized as potential indicators of lung cancer in serum or plasma, including haptoglobin (HP), inter-alpha-trypsin inhibitor heavy chain 4 (ITI-H4), complement C3 precursor, and leucine-rich alpha-2-glycoprotein. In addition, plasma kallikrein (KLKB1) and inter-alpha-trypsin inhibitor heavy chain 3 (ITI-H3) were identified as being potentially elevated in the lung cancer group, and were validated by Western blot analysis. Furthermore, approximately 18 kDa plasma kallirein protein fragment was detected at high levels in 25 out of 28 adenocarcinoma patients, while one of the eight normal individuals showed moderate positive. The results suggest that KLKB1 represents a potential candidate serum biomarker of lung cancer.
165 citations
TL;DR: QconCATs offer a new and efficient approach to precise and simultaneous absolute quantification of multiple proteins, subproteomes, or even entire proteomes.
162 citations
TL;DR: The development of a robust and relatively high-throughput method for in-depth proteomic analysis of human plasma suitable for biomarker discovery and that identifies proteins that may be of particular interest when studying a variety of disease conditions is reported to be present in normal plasma.
Abstract: We report on the development of a robust and relatively high-throughput method for in-depth proteomic analysis of human plasma suitable for biomarker discovery. The method consists of depletion of albumin and IgG and multi-lectin affinity chromatography (M-LAC), followed by nanoLC-MS/MS analysis of digested proteins and label-free comparative quantitation of proteins. The performance of the method is monitored by multiple quality control points to ensure reproducibility of the analysis. The method identifies proteins that are reported to be present in normal plasma at concentrations of 10−100 ng/mL and that may be of particular interest when studying a variety of disease conditions. Numerous tissue leakage proteins of potentially even lower concentrations are also identified. When the method was used in a study to identify potential biomarkers of psoriasis, the differential abundance of proteins present at low μg/mL level was quantitated and later verified by ELISA measurements. Keywords: plasma • multi-l...
131 citations
TL;DR: Immobilized metal‐ion affinity chromatography (IMAC) represents a relatively new separation technique that is primarily appropriate for the purification of proteins with natural surface‐exposed histidine residues and for recombinant proteins with engineered histidine tags or histidine clusters.
Abstract: The correct folding of solubilized recombinant proteins is of key importance for their production in industry. On‐column refolding of proteins is mainly achieved by three methods: size‐exclusion chromatography, ion exchange chromatography and affinity chromatography using immobilized metal chelates. The principles of these methods were first laid down in the 1990s, but many recent improvements have been made to these processes. Immobilized metal‐ion affinity chromatography (IMAC) represents a relatively new separation technique that is primarily appropriate for the purification of proteins with natural surface‐exposed histidine residues and for recombinant proteins with engineered histidine tags or histidine clusters. Because the method has gained broad popularity in recent years, the main recent developments in the field of new sorbents, techniques and possible applications are discussed in this article.
130 citations
TL;DR: Four Escherichia coli expression vectors are constructed that can be used for ligation independent cloning (LIC) of an amplified target gene sequence and the resultant recombinant SUMO fusion proteins can be overexpressed in E. coli and readily isolated by standard affinity purification techniques.
125 citations
TL;DR: Investigation of the production of a genetic fusion with a glycine-rich homo-amino-acid polymer (HAP) as an alternative way to attach a solvated random chain with large hydrodynamic volume found it to be beneficial for specialized medical applications.
Abstract: Chemical conjugation of small recombinant proteins with polyethylene glycol (PEG) is an established strategy to extend their typically short circulation times to a therapeutically useful range. We have investigated the production of a genetic fusion with a glycine-rich homo-amino-acid polymer (HAP) as an alternative way to attach a solvated random chain with large hydrodynamic volume. The anti-HER2 Fab fragment 4D5 was used as a model system and fused with either 100 or 200 residue polymers of the repetitive sequence (Gly(4)Ser)(n) to its light chain. Both fusion proteins were successfully produced in the periplasm of Escherichia coli and obtained as homogeneous preparations after two-step affinity chromatography via the His(6) tag fused to the heavy chain and the Strep-tag II fused to the extended light chain. Both modified Fab fragments showed binding activity towards the HER2 antigen indistinguishable from the conventional recombinant Fab fragment. When compared with the unfused Fab fragment, a significantly increased hydrodynamic volume, by ca. 120%, was observed during gel filtration for the 200 residue HAP fusion protein and, to a lesser extent, in the case of the 100 residue HAP. Difference CD measurements revealed a characteristic random coil spectrum for the 100 and 200 residue HAP fusion moieties. Finally, pharmacokinetic experiments were carried out in mice after radioiodination of the recombinant Fab fragments. Although the 100 residue HAP fusion showed a behavior very similar to the unfused Fab fragment, with a terminal plasma half-life of ca. 2 h, the 200 residue HAPylated Fab fragment gave rise to a significantly prolonged half-life of ca. 6 h. While this moderate effect may so far be most beneficial for specialized medical applications, such as in vivo imaging, the genetic engineering of optimized HAP sequences should yield pharmacokinetic properties similar to PEGylation, yet without necessitating in vitro modification steps.
122 citations
TL;DR: N-glycosylated proteins were isolated from Arabidopsis thaliana mature stems using affinity chromatography on Concanavalin A Sepharose, separated by 2D-electrophoresis and identified using nanoHPLC-MS/MS and MALDI-TOF MS.
Abstract: N-glycosylated proteins were isolated from Arabidopsis thaliana mature stems using affinity chromatography on Concanavalin A Sepharose, separated by 2D-electrophoresis and identified using nanoHPLC-MS/MS and MALDI-TOF MS. 102 glycoproteins were identified. 94% of these proteins were predicted by bioinformatics to be targeted to the secretory pathway and 87% of them were predicted to be localized in the cell wall or at the plasma membrane. 30% of these proteins belong to glycoside hydrolase (GH) families with some of them possibly involved in the hydrolysis of cell wall polysaccharides. The second major class of identified proteins comprises aspartyl and serine proteases. Other proteins are predicted to be oxido-reductases, contain interacting domains, are potentially involved in signalling or have an unknown function. This is, to our knowledge, the first survey of plant cell wall N-glycosylated proteins.
121 citations
TL;DR: Elution from the Protein A resin was found to increase the aggregation rate constants over and above what would be expected from exposure to low pH conditions in solution alone, demonstrating that Protein A-Fc interactions can destabilize product structure and increase the tendency to aggregate.
TL;DR: This work describes the standard protocol using an automated FAC system (FAC-1), consisting of two isocratic pumps, an autosampler, a column oven and two miniature columns connected to a fluorescence detector, which has so far obtained quantitative interaction data of >100 lectins for >100 PA oligosaccharides.
Abstract: Frontal affinity chromatography using fluorescence detection (FAC-FD) is a versatile technique for the precise determination of dissociation constants (Kd) between glycan-binding proteins (lectins) and fluorescent-labeled glycans. A series of glycan-containing solutions is applied to a lectin-immobilized column, and the elution profile of each glycan (termed the 'elution front', V) is compared with that (V0) for an appropriate control. Here we describe our standard protocol using an automated FAC system (FAC-1), consisting of two isocratic pumps, an autosampler, a column oven and two miniature columns connected to a fluorescence detector. Analysis time for 100 sugar-protein interactions is approximately 10 h, using as little as 2.5 pmol of pyridylaminated (PA) oligosaccharide per analysis. Using FAC-FD, we have so far obtained quantitative interaction data of >100 lectins for >100 PA oligosaccharides.
TL;DR: The results demonstrate the utility of PQD operation on the LTQ instrument for quantitative analysis of iTRAQ reagent‐labeled peptide mixtures, as well as the quantitative reproducibility of the avidin‐affinity enrichment method.
Abstract: We describe a strategy for the identification of carbonylated proteins from complex protein mixtures that combines biotin hydrazide labeling of protein carbonyl groups, avidin affinity chromatography, multiplexed iTRAQ reagent stable isotope labeling, and analysis using pulsed Q dissociation (PQD) operation on an LTQ linear ion trap mass spectrometer. This strategy provided the ability to distinguish biotin hydrazide labeled, avidin purified, carbonylated proteins from non-carbonylated background proteins with affinity for the avidin column, derived from a control sample. Applying this strategy to the identification of crudely enriched rat skeletal muscle mitochondrial protein isolates, we generated a catalogue of over 200 carbonylated proteins by virtue of their quantitative enrichment compared to the control sample. The catalogue contains many mitochondrial localized proteins shown to be susceptible to carbonyl modification for the first time, including numerous transmembrane proteins involved in oxidative phosphorylation. Other oxidative modifications (e.g. nitrosylation, hydroxylation) were also identified on many of the carbonylated proteins, providing further evidence of the susceptibility of these proteins to oxidative damage. The results also demonstrate the utility of PQD operation on the LTQ instrument for quantitative analysis of iTRAQ reagent-labeled peptide mixtures, as well as the quantitative reproducibility of the avidin-affinity enrichment method.
TL;DR: Erythrina lectins can serve as effective substitutes for RCA120, taking the above differences into consideration.
Abstract: Ricinus communis agglutinin I (RCA120) is considered a versatile tool for the detection of galactose-containing oligosaccharides However, possible contamination by the highly toxic isolectin 'ricin' has become a critical issue for RCA120's continued use From a practical viewpoint, it is necessary to find an effective substitute for RCA120 For this purpose, we examined by means of frontal affinity chromatography over 100 lectins which have similar sugar-binding specificities to that of RCA120 It was found that Erythrina cristagalli lectin (ECL) showed the closest similarity to RCA120 Both lectins prefer Gal beta1-4GlcNAc (type II) to Gal beta1-3GlcNAc (type I) structures, with increased affinity for highly branched N-acetyllactosamine-containing N-glycans Their binding strength significantly decreased following modification of the 3-OH, 4-OH and 6-OH of the galactose moiety of the disaccharide, as well as the 3-OH of its N-acetylglucosamine residue Several differences were also observed in the affinity of the two lectins for various other ligands, as well as effects of bisecting GlcNAc and terminal sialylation Although six other Erythrina-derived lectins have been reported with different amino acid sequences, all showed quite similar profiles to that of ECL, and thus, to RCA120 Erythrina lectins can therefore serve as effective substitutes for RCA120, taking the above differences into consideration
TL;DR: It is indicated that four outer membrane proteins of V. parahaemolyticus zj2003, including OmpW, OMPV, OmpU and OmpK, are immunogenic during in vivo infection, which would be of some significance in developing efficient vaccine in aquaculture.
TL;DR: The procedure described in this study will provide a reliable and simple method for production of different cationic peptides for biological studies and demonstrate that recombinant Mdmcec had a broad spectrum of activity against fungi, Gram-positive and negative bacteria.
TL;DR: This work shows that affinity chromatography can be used for the purification of copper-chelating peptides and probably other metals of nutritional interest such as calcium, iron, and zinc.
Abstract: Chickpea protein hydrolysates obtained with alcalase and flavourzyme were used for purification of copper chelating peptides by affinity chromatography using copper immobilized on solid supports. The chelating activity of purified peptides was indirectly measured by the inhibition of beta-carotene oxidation in the presence of copper. Two protein hydrolysates, obtained after 10 and 100 min of hydrolysis, were the most inhibitory of beta-carotene oxidation. Purified copper chelating peptides from these protein hydrolysates contained 19.7 and 35.1% histidine, respectively, in comparison to 2.7 and 2.6% in the protein hydrolysates. Chelating peptides from hydrolysate obtained after 10 min of hydrolysis were the most antioxidative being 8.3 times more antioxidative than the hydrolysate, while chelating peptides purified from protein hydrolysate obtained after 100 min were 3.1 times more antioxidative than its hydrolysate. However, the histidine content was higher in peptides derived from the 100 min hydrolysate (19.7 against 35.1% in 10 min hydrolysate), indicating that this amino acid is not the only factor involved in the antioxidative activity, and other factors such as peptide size or amino acid sequence are also determinant. This manuscript shows that affinity chromatography is a useful procedure for purification of copper chelating peptides. This method can be extended to other metals of interest in nutrition, such as calcium, iron, or zinc. Purified chelating peptides, in addition to their antioxidative properties, may also be useful in food mineral fortification for increasing the bioavailability of these metals.
TL;DR: The enzyme showed no specific requirements for metal ions, with the exception of Mg2+, which enhanced both activity and stability, and was used for the synthesis of prebiotic galacto-oligosaccharides (GOS) from lactose.
Abstract: Beta-galactosidase from the probiotic strain Lactobacillus acidophilus R22 was purified to apparent homogeneity by ammonium sulphate fractionation, hydrophobic interaction, and affinity chromatography. The enzyme is a heterodimer consisting of two subunits of 35 and 72 kDa, as determined by gel electrophoresis. The optimum temperature of beta-galactosidase activity was 55 degrees C (10-min assay) and the range of pH 6.5-8, respectively, for both o-nitrophenyl-beta-D-galactopyranoside (oNPG) and lactose hydrolysis. The Km and Vmax values for lactose and oNPG were 4.04+/-0.26 mM, 28.8+/-0.2 micromol D-glucose released per min per mg protein, and 0.73+/-0.07 mM, 361+/-12 micromol o-nitrophenol released per min per mg protein, respectively. The enzyme was inhibited by high concentrations of oNPG with Ki,s=31.7+/-3.5 mM. The enzyme showed no specific requirements for metal ions, with the exception of Mg2+, which enhanced both activity and stability. The genes encoding this heterodimeric enzyme, lacL and lacM, were cloned, and compared with other beta-galactosidases from lactobacilli. Beta-galactosidase from L. acidophilus was used for the synthesis of prebiotic galacto-oligosaccharides (GOS) from lactose, with the maximum GOS yield of 38.5% of total sugars at about 75% lactose conversion.
TL;DR: Comparable performance was observed for the virus‐based polyclonal versus a commercial monoclonal antibody raised against the globo‐H tetrasaccharide; this highlights the utility of the glycan microarray for both quality control and rapid in‐depth analysis.
Abstract: Tetra- and hexasaccharides were arrayed on the exterior surface of cowpea mosaic virus by using a copper-catalyzed azide–alkyne cycloaddition reaction. Inoculation of chickens with these virus conjugates gave rise to large quantities of polyclonal anti-glycan IgY antibodies that displayed excellent avidity and specificity on analysis with printed glycan microarrays. Avian IgY antibodies are produced in significantly higher yield than is possible for mouse or rabbit IgG, and exhibit reduced cross reactivity with native mammalian proteins. For a tri-LacNAc antigen, affinity purification against immobilized mono-LacNAc was necessary to provide a set of antibodies with specific binding properties. Comparable performance was observed for the virus-based polyclonal versus a commercial monoclonal antibody raised against the globo-H tetrasaccharide; this highlights the utility of the glycan microarray for both quality control and rapid in-depth analysis. Virus–carbohydrate conjugates are promising candidates for development in diagnostic and immunotherapeutic applications.
TL;DR: The results suggested that PjLec may be an important humoral defence factor against bacterial infection in P. japonicus.
TL;DR: The presented study aims on the development of a capture step for the purification of cell culture derived influenza viruses using lectin affinity chromatography using human influenza A/Puerto Rico/8/34 virus produced in Madin Darby canine kidney cells as a model.
TL;DR: It is found that intact protein A leaches when loading either purified antibody or unpurified antibody in harvested cell culture fluid (HCCF), and that additionally fragments ofprotein A leach when loading HCCF, suggesting that proteinases contribute to the generation of protein A fragments.
TL;DR: To determine the ratio of thiols existing as mixed disulfides following oxidation, a method combining fast purification of albumin using affinity columns and high-performance liquid chromatography with fluorescence detection for low molecular weight thiolS which were labeled after reduction was developed.
TL;DR: This article gives an overview of recently reported strategies in lectin immobilization to different stationary phases in chromatography and electrophoresis in order to support lectin-affinity-interaction-based bioseparation methodologies.
Abstract: Given the high specificity of plant lectins, it is not surprising that their immobilized forms are more and more frequently used for affinity-based analysis of glycoproteins. Common lectin-immobilization techniques range from reversible, non-covalent attachments to covalent immobilization onto a vast palette of substrates (e.g., agarose, silica, and polymeric materials). This article gives an overview of recently reported strategies in lectin immobilization to different stationary phases in chromatography and electrophoresis in order to support lectin-affinity-interaction-based bioseparation methodologies.
TL;DR: This study identifies SSAP as a new member of the LAO family and reveals LAO involvement in the innate immunity of fish skin.
Abstract: Fish skin mucus contains a variety of antimicrobial proteins and peptides that seem to play a role in self defense. We previously reported an antibacterial protein in the skin secretion of the rockfish, Sebastes schlegeli, which showed selective antibacterial activity against Gram-negative bacteria. This study aimed to isolate and structurally and functionally characterize this protein. The antibacterial protein, termed SSAP (S. schlegeli antibacterial protein), was purified to homogeneity by lectin affinity column chromatography, anion-exchange HPLC and hydroxyapatite HPLC. It was found to be a glycoprotein containing N-linked glycochains and FAD. Its molecular mass was estimated to be 120 kDa by gel filtration HPLC and 53 kDa by SDS/PAGE, suggesting that it is a homodimer. On the basis of the partial amino-acid sequence determined, a full-length cDNA of 2037 bp including an ORF of 1662 bp that encodes 554 amino-acid residues was cloned by 3′ RACE, 5′ RACE and RT-PCR. A blast search showed that a mature protein (496 residues) is homologous to l-amino acid oxidase (LAO) family proteins. SSAP was determined to have LAO activity by the H2O2-generation assay and substrate specificity for only l-Lys with a Km of 0.19 mm. It showed potent antibacterial activity against fish pathogens such as Aeromonas hydrophila, Aeromonas salmonicida and Photobacterium damselae ssp. piscicida. The antibacterial activity was completely lost on the addition of catalase, confirming that H2O2 is responsible for the growth inhibition. This study identifies SSAP as a new member of the LAO family and reveals LAO involvement in the innate immunity of fish skin.
TL;DR: The purified preparation of GAD from rice germ indicated that the GAD existed as a dimer of homological subunits, and Chemicals reagents such as HgCl2, KI and AgNO3 decreased the enzyme activity, but 500 μM of CaCl2 at the optimum pH could increase the activity by 145%.
TL;DR: The use of high-performance lectin affinity enrichment of glycoproteins at microscale levels using a series of silica-bound lectins for glycoprotein enrichment from microliter volumes of human blood serum shows substantial reduction of sample losses.
Patent•
04 Sep 2007TL;DR: In this paper, the authors provided novel compositions derived from antigen-binding sites of immunoglobulins having affinity for IL-31, which are useful in specific binding assays, affinity purification schemes, drug or toxin targeting, imaging, and genetic or immunological therapeutics for inflammatory diseases.
Abstract: Novel compositions derived from antigen-binding sites of immunoglobulins having affinity for IL-31 are provided. The compositions exhibit immunological binding properties of antibody molecules capable of binding specifically to a human IL-31. CDR regions derived from same or different immunoglobulin moieties are provided. Also provided are single chain polypeptides wherein VH and VL domains are attached. The sFv molecules can include ancillary polypeptide moieties which can be bioactive, or which provide a site of attachment for other useful moieties. The compositions are useful in specific binding assays, affinity purification schemes, drug or toxin targeting, imaging, and genetic or immunological therapeutics for inflammatory diseases. The invention thus provides novel polypeptides, the DNAs encoding those polypeptides, expression cassettes comprising those DNAs, and methods of inducing the production of the polypeptides. The invention further provides the amino acid sequences of the variable regions of the monoclonal antibodies and use of these monoclonal antibody or antibody fragment in conjunction with an an human IgG4 Fc molecule.
TL;DR: A rapid and efficient method for the purification of CK1 by affinity chromatography on an immobilised fragment of axin, which was found to selectively bind native CK1alpha and CK1epsilon from porcine brain and displayed high kinase activity.
TL;DR: The effects of oxidative stress on the yeast proteome were studied using hydrogen peroxide as the stress agent and it was concluded that the position of the peptides identified in the primary structure of protein candidates derived from databases occurred by fragmentation of a parent protein.