Crude Protein Mixtures Research Articles

Affinity Chromatography of Tryptases: Design, Synthesis and Characterization of a Novel Matrix‐Bound Bivalent Inhibitor

beta-Tryptases are mast cell-derived serine proteases that are enzymatically active in the form of an oligomer consisting of four subunits each with trypsin-like activity. The active-site clefts, which are directed toward the central pore of the tetramer, form spatial arrays of four negatively charged S1 binding pockets. Therefore, dibasic inhibitors of appropriate geometry can bind in a bivalent fashion to neighboring subunits. We have recently identified a potent bivalent inhibitor (K(i)=18 nM), based on the bifunctional scaffold cyclo-(-D-Asp-L-Asp-) and the arginine mimetic dl-3-aminomethyl-phenylalanine methyl ester as a ligand for S1 pockets that takes advantage of the this unique tetrameric geometry. To generate an affinity matrix, the bivalent ligand was modified and immobilized on a Sepharose matrix by use of the PEG derivative Jeffamine ED 900 as spacer. This matrix selectively recognizes and binds beta-tryptase from crude protein mixtures and thus is useful as a geometry-driven means of isolating and purifying human mast cell tryptases.

A proteomic approach to characterize protein shedding

Shedding (i.e. proteolysis of ectodomains of membrane proteins) plays an important pathophysiological role. In order to study the feasibility of identifying shed proteins, we analyzed serum-free media of human mammary epithelial cells by mass spectrometry following induction of shedding by the phorbol ester, 4 beta-phorbol 12-myristate 13-acetate (PMA). Different means of sample preparation, including biotinylation of cell surface proteins, isolation of glycosylated proteins, and preparation of crude protein fractions, were carried out to develop the optimal method of sample processing. The collected proteins were digested with trypsin and analyzed by reversed-phase capillary liquid chromatography interfaced to an ion-trap mass spectrometer. The resulting peptide spectra were interpreted using the program SEQUEST. Analyzing the sample containing the crude protein mixture without chemical modification or separation resulted in the greatest number of identifications, including putatively shed proteins. Overall, 45 membrane-associated proteins were identified including 22 that contain at least one transmembrane domain and 23 that indirectly associate with the extracellular surface of the plasma membrane. Of the 22 transmembrane proteins, 18 were identified by extracellular peptides providing strong evidence they originate from regulated proteolysis or shedding processes. We combined results from the different experiments and used a peptide count method to estimate changes in protein abundance. Using this approach, we identified two proteins, syndecan-4 and hepatoma-derived growth factor, whose abundances increased in media of cells treated with PMA. We also detected proteins whose abundances decreased after PMA treatment such as 78 kDa glucose-regulated protein and lactate dehydrogenase A. Further analysis using immunoblotting validated the abundance changes for syndecan-4 and 78 kDa glucose-regulated protein as a result of PMA treatment. These results demonstrate that tandem mass spectrometry can be used to identify shed proteins and to estimate changes in protein abundance.

Chemical modification of chicken egg white proteins with polyethyleneoxide-maleic anhydride copolymer and their partitioning in aqueous two-phase systems

Aqueous two-phase partitioning is one of the excellent bioseparation systems for proteins and other biomolecules in water-rich solutions containing polymers or salts. To improve the partition properties of model proteins, chicken egg white (CEW) proteins were chemically modified with polyethyleneoxide–maleic anhydride (PEOMA) copolymer. The modified proteins were partitioned in polyethyleneoxide–maleic acid/potassium phosphate aqueous two-phase systems. Firstly, the contribution of the chemical modification of proteins to the partition coefficient was investigated by using modified proteins with different degrees of modification (DM). The partition coefficients of modified proteins increased with increasing DM. The results suggested that the surface of the modified proteins had a greater hydrophobic property, similar to the PEOMA copolymer. Secondly, the effect of the pH on the partitioning of the modified proteins was studied. The partitioning of the modified proteins was found to be more dependent on pH in the alkaline systems. The increasing partition coefficient with pH at alkaline pH can be explained by the electrostatic repulsion between the phosphate anions and the maleic groups attached to the proteins. These results suggested that chemical modification of proteins with PEOMA could be a useful tool for separation and purification of crude protein mixtures. Copyright © 2005 Society of Chemical Industry

Isolation of organelles and prefractionation of protein extracts using free-flow electrophoresis.

One of the major obstacles in the analysis of proteomes is the extreme complexity of any particular cell or biological fluid. Free-flow electrophoresis (FFE) is a powerful tool for reduction of this complexity, which is a prerequisite for systematic and comprehensive protein analyses. Protocols are provided in this unit for sample fractionation at two different stages: on the protein level by isoelectric focusing FFE fractionation of crude protein mixtures such as whole cell lysates, and on a subcellular level by zone-electrophoretic FFE purification of organelles.

A top down approach to protein structural studies using chemical cross-linking and Fourier transform mass spectrometry.

Mass spectrometric analysis of wild-type proteins that have been covalently modified by bifunctional cross-linking reagents and then digested proteolytically can be used to obtain low-resolution distance constraints, which can be useful for protein structure determination. Limitations of this approach include time-consuming separation steps, such as the separation of internally cross-linked protein monomers from covalent dimers, and a susceptibility to artifacts due to low levels of natural and man-made peptide modifications that can be mistaken for cross-linked species. The results presented here show that when a crude cross-linked protein mixture is injected into an electrospray ionization Fourier transform mass spectrometry (ESI-FTMS) instrument, the cross-link positions can be localized by fragmentation and mass spectrometry on the 'gas-phase purified' singly internally cross-linked monomer. Our results show that reaction of ubiquitin with the homobifunctional lysine-lysine cross-linking reagent dissuccinimidyl suberate (DSS) resulted in two cross-links consistent with the known ubiquitin tertiary structure (K6-K11 and K48-K63). Because no protein or peptide chemistry steps are needed, other than the initial cross-linking, this new top down approach appears well suited for high-throughput experiments with multiple cross-linkers and reaction conditions. Published in 2002 by John Wiley & Sons, Ltd.

Semi-continuous protein fractionating using Affinity Cross-Flow Filtration

Protein purification by means of downstream processing is increasingly important. At the University of Twente a semi-continuous process is developed for the isolation of BSA out of crude protein mixtures. For this purpose an automated Affinity Cross-Flow Filtration, ACFF, process is developed. This process combines the advantages of both affinity adsorption, having high resolution, and microfiltration, for rapid and large continuous processing capacity. The overall process contains 4 sequential steps: affinity binding; purification; dissociation; regeneration. In the first step binding of the desired protein to a tailor-made ligand-immobilized particle is established. This temporary fixation of target proteins on a large entity allows us to wash out the unbound components in the purification step using high flux microfiltration membranes. After washing out the unbound proteins the target protein-ligand immobilized particle complex is dissociated by adding an appropriate salt. In a second filtration cycle the purified protein can be collected as the permeate. In the last step the ligand-immobilized particle is regenerated for the next cycle with buffer. Using this process it was possible to obtain the target protein BSA with a purity of more than 95%.

Epoxide electrophiles as activity-dependent cysteine protease profiling and discovery tools

Background: Analysis of global changes in gene transcription and translation by systems-based genomics and proteomics approaches provides only indirect information about protein function. In many cases, enzymatic activity fails to correlate with transcription or translation levels. Therefore, a direct method for broadly determining activities of an entire class of enzymes on a genome-wide scale would be of great utility. Results: We have engineered chemical probes that can be used to broadly track activity of cysteine proteases. The structure of the general cysteine protease inhibitor E-64 was used as a scaffold. Analogs were synthesized by varying the core peptide recognition portion while adding affinity tags (biotin and radio-iodine) at distal sites. The resulting probes containing a P2 leucine residue (DCG-03 and DCG-04) targeted the same broad set of cysteine proteases as E-64 and were used to profile these proteases during the progression of a normal skin cell to a carcinoma. A library of DCG-04 derivatives was constructed in which the leucine residue was replaced with all natural amino acids. This library was used to obtain inhibitor activity profiles for multiple protease targets in crude cellular extracts. Finally, the affinity tag of DCG-04 allowed purification of modified proteases and identification by mass spectrometry. Conclusions: We have created a simple and flexible method for functionally identifying cysteine proteases while simultaneously tracking their relative activity levels in crude protein mixtures. These probes were used to determine relative activities of multiple proteases throughout a defined model system for cancer progression. Furthermore, information obtained from libraries of affinity probes provides a rapid method for obtaining detailed functional information without the need for prior purification/identification of targets.

Adsorption and selectivity characteristics of several human serum proteins with immobilised hard Lewis metal ion–chelate adsorbents

In this investigation, human serum has been used as an example of a crude protein mixture to define the protein binding characteristics and selectivity of several immobilised hard Lewis metal ion affinity chromatographic (IMAC) adsorbents. Specifically, the binding properties of immobilised O-phosphoserine ( im-OPS) and 8-hydroxyquinoline ( im-8-HQ), with immobilised iminodiacetic acid as a control system, have been investigated in combination with the hard Lewis metal ions, Al 3+, Ca 2+, Fe 3+, Yb 3+, and the borderline metal ion, Cu 2+, over the pH range pH 5.5 to pH 8.0 with buffers of 0.5 M ionic strength. The same IMAC adsorbents were also investigated for their protein binding capabilities with buffers of an ionic strength of 0.06 M at pH 5.5 and pH 8.0. The binding behaviour of four “marker” proteins, namely transferrin, α 2-macroglobulin, gammaglobulin and human serum albumin have furthermore been employed to monitor the differences in protein selectivity exhibited by these IMAC systems. The experimental findings confirm that these hard Lewis metal ion IMAC adsorbents function in a “mixed” binding mode with both coordination and electrostatic characteristics evident, depending on the ionic strength and pH of the equilibration or elution buffers. Based on a screening protocol, several members of the im-M n+ –8-HQ and im-M n+ –OPS adsorbent series have been identified with high selectivity for transferrin and α 2-macroglobulin. These hard Lewis metal ion IMAC adsorbents thus provide attractive alternatives for selective fractionation of human serum proteins.

The Functional Role of a DNA Primase in Chloroplast DNA Replication in Chlamydomonas reinhardtii

A complementation experiment was developed to identify the protein component that is essential for the in vitro replication of a cloned template containing a chloroplast DNA replication origin of Chlamydomonas reinhardtii. Using this method, we have identified a DNA primase activity that copurified with DNA polymerase from the crude protein mixture. The primase catalyzed the synthesis of short RNA primers on single-stranded DNA templates. Among the synthetic templates, the order of preference was poly(dA), poly(dT), and poly(dC). The primer size range for these templates was 11–18, 5–12, and 3–11 nucleotides, respectively. On a single-stranded template containing the chloroplast DNA replication origin, the primer length range reached 19 to 27 nucleotides, indicating a better processtivity. Several initiation sites were mapped on both strands of the cloned replication origin. Some preferential initiation sites were located on A tracks spaced at one helical turn apart within the bending locus. Primase improved the template specificity of the in vitro DNA replication system and enhanced the incorporation of radioactive dATP into the supercoiled template containing the core sequences of the chloroplast DNA replication origin.

床皮飼料を摂取したニワトリの消化率と窒素蓄積率

Digestibilities and nitrogen retention of non-gelatinized and gelatinized limed splits, and effects of the limed splits on digestibilities of nutrients were studied in colostomized chickens. Egg albumen (control diet), a 1:1 (on the basis of crude protein) mixture of egg albumen and non -gelatinized limed split (AS diet) and that of egg albumen and gelatinized limed split (AGS diet) were used as the dietary protein source. All diets contained 14% crude protein. Decreased feed consumption was observed in AS diet and AGS diet groups. There was no difference in digestibilities of nutrients among 3 groups, but nitrogen retention in AS and AGS diet groups was lower than in control group. Results obtained here indicate that more than 90% of limed split is digested by chickens irrespective of gelatinization and that the palatability of limed split should be improved for practical use.

床皮飼料を強制給餌したニワトリヒナの成長と窒素利用性

The aim of the present study is to estimate utilization of limed split as a protein source of the diet for chickens. Egg albumen (control diet), a 1:1 (on the basis of crude protein) mixture of egg albumen and limed split (AS diet) and that of egg albumen and gelatinized limed split (AGS diet) were used as the dietary protein source. All diets contained 20% of crude protein. Chicks were force-fed on the diets for 10 days. Composition and contents of amino acids in limed split were much the same as those in gelatinized one. Out of essential amino acids, glycine was extremely excessive, while histidine, isoleucine, methionine and tyrosine were deficient, and cystine and tryptophan were negligible. AS and AGS diets were suboptimal in threonine and tryptophan. There was no significant difference in body weight gain, feed efficiency and nitrogen retention among control, AS and AGS groups. Results obtained here suggest that limed split may be used as a protein source of the diet for chicks, supplying essential amino acids, and that gelatinization of limed split may be unnecessary.

Synthesis and characterization of a carbene-generating biotinylated N-acetylglucosamine for photoaffinity labeling of β-(1 → 4)-galactosyltransferase

A photoreactive N-acetylglucosamine derivative, N-[2-[2-[2-(2-biotinylaminoethoxy)-ethoxy]ethoxy]-4-[3-(trifluoromethyl)-3 H- diazirin-3-yl]benzoyl]-N 4-[2- (acetylamino)-2-deoxy-β- d-glucopyranosyl]- l-aspartamide (BDGA), was synthesized as a carbene-generating biotinylated probe for UDP-galactose: N-acetylglucosamine β-(1 → 4)-galactosyltransferase (GalT). The photoaffinity labeling experiments of bovine GalT with BDGA under various conditions were examined based on the quantitative chemiluminescent detection of the biotinyl residue which was photochemically introduced into the GalT protein. A progressive decrease in the yield of specific photolabeling was observed upon lowering the incubation temperature from 37 °C to 20 °C or 4 °C. The amount of photoincorporation was also decreased when UMP was not included in the incubation mixture. Using a crude protein mixture of recombinant human GalT, a band corresponding to the glutathione S-transferase fusion GalT protein was also specifically visualized. Furthermore, combined use of BDGA photolabeling with an immobilized avidin was found to be effective for the selective retrieval of photolabeled GalT from a reaction mixture containing a large amount of unlabeled GalT protein. The results obtained clearly demonstrate that the covalent biotinylation using the carbene-generating photoaffinity reagent BDGA would be useful for the analysis of acceptor substrate binding sites within the GalT protein.

Capillary zone electrophoresis of proteins with hydrophilic fused-silica capillaries

Fused-silica capillaries having surface-bound hydroxylated polyether functions were developed for the separation of proteins by capillary zone electrophoresis. In one approach, the hydrophilic coatings consisted of two layers: a glyceropropylpolysiloxane sublayer covalently attached to the inner surface and a polyether top layer. In a second approach, the capillary wall was coated with polysiloxane polyether chains the monomeric units of which at both ends were covalently attached to the capillary inner surface with possible interconnection. These coatings yielded capillaries with different electroosmotic flow characteristics. The relatively long polyether chains of the various coatings were effective in shielding the unreacted surface silanols, thus minimizing solute-wall adsorption. As a consequence, high separation efficiencies were obtained in the pH range 4.0–7.5, which allowed the separation of widely differing proteins, the characterization of heterogeneous proteins and the fingerprinting of crude protein mixtures. The various coatings were stable and exhibited reproducible separations from run-to-run, day-to-day, and column-to-column. Furthermore, a procedure was developed for restoring collapsed capillaries after prolonged use.

Application of Reverse Micelles for the Extraction of Amino Acids and Proteins

The use of water-in-oil microemulsions for the solubilization and extraction of amino acids and proteins is investigated, using both the liquid-liquid extraction and the solid-liquid extraction method. The solubilization behaviour of small monomeric proteins such as cytochrome c, lysozyme, ribonuclease A, trypsin, ?-chymotrypsin, papain, pepsin, and bovine serum albumin (BSA) into mainly AOT (the anionic surfactant bis(2-ethylhexyl) sodium sulfosuccinate)/H2O/isooctane is studied by varying several external parameters, such as wo (wo = [H2O]/[AOT]), surfactant type and concentration in the organic phase, and, pH, ionic strength, salt type in the H2O phase. In the liquid-liquid extraction method, the present biphasic system consists of an aqueous and a supernatant reverse micellar solution. The compounds are transferred from the H2O phase into the organic micellar phase. This process is investigated also in view of the simultaneously occurring H2O transfer. Studies of transfer kinetics give an information about the connection between the H2O and protein extraction. In the second technique, the compounds are given in their solid state and suspended in the organic micellar solution which contains already a known amount of solubilized water.With the liquid-liquid phase-transfer method, it is shown that, under certain conditions, a quantitative and quite remarkable transfer can be achieved, whereby the salt type and its concentration in the H2O phase, as well as the aqueous pH, play the most important role in determining the extent mass transfer. The significance of electrostatic interactions between the micelles and the extracted molecules is evidenced also by comparing extraction results obtained with AOT and CTAB (cetyltrimethylammonium bromide; is a cationic surfactant) water-in-oil microemulsions. Moreover, the extraction yield of a series of positively, negatively charged, and neutral amino-acid derivatives is investigated in the two surfactant systems, in order to characterize the significance of electrostatic and hydrophobic interactions with the surfactant molecules. This series of amino-acid derivatives is also studied with the solid-phase extraction method. In the case of proteins transferred from the solid state, the solubilization yield is affected by the protein and micellar droplet size. For small proteins, i.e. with a molecular weight below ca. 15 000 D, the extraction curve as a function of wo shows surprisingly enough a peak in the small wo region (between 6 and 12). By increasing the H2O content above this wo limit, the transfer efficiency of the H2O-soluble proteins decreases. For proteins having a molecular weight above 20 000 D, the extraction curve as a function of wo shows a monotonous increase with increasing wo without any sharp wo maxima. A qualitative model is offered to explain this different behaviour. It is also shown that the ionization state of commercially available protein powders markedly affects the extraction behaviour. The micellar exclusion effect is finally applied in the extraction of proteins from a crude bacterial protein mixture.

Simultaneous ultrafiltration and affinity sorptive separation of proteins in a hollow fiber membrane module

A protein separation scheme combining affinity or ion exchange sorption with hollow fiber cross-flow filtration is described. Sorptive gel particles were loaded into the shell side of a hollow fiber membrane module. In the adsorption step, crude protein mixtures were passed through the lumen and permeating proteins passed through the membrane to bind on the gel particles in the shell. During elution, a buffer of adequate ionic strength to desorb the bound proteins was passed through the lumen and permeated through the shell. The eluant was then collected at the outlet to the shell of the hollow fiber module. The concept is illustrated by two examples: the purification of butyrylcholinesterase (EC 3.1.1.7) from raw horse serum using the affinity gel procainamide-Sepharose as the packing and the separation of carboxylesterase (EC 3.1.1.1) from beef liver homogenate using DEAE-Sephadex as the packing. The technique has the advantage of high volumetric throughputs typical of hollow fiber membrane modules as well as the high capacity characteristic of chromatographic packings. In addition, cross-flow filtration of particulates, agglomerates, and debris in passing protein from lumen to shell side can help eliminate the need for extensive pretreatment.

Analytic and preparative separation of apolipoproteins A-I, A-II, and A-IV by reverse phase high pressure liquid chromatography

We have developed a rapid reverse phase high pressure liquid chromatographic technique capable of separating apolipoproteins A-I, A-II, and A-IV and their constituent isoforms. The separations were performed on a 30 cm x 3.9 mm C18 reverse-phase silica-based column (Waters Associates) using a 47-55% gradient of acetonitrile in 0.1% trifluoroacetic acid. Injection of a mixture of equal weights of apolipoproteins A-I, A-II, and A-IV yielded a single peak for apoA-IV, two peaks of apoA-I isoforms, and three peaks of apoA-II isoforms, with negligible overlap of each peak. When applied to the isolation of apoA-IV from a crude protein mixture obtained by incubation of phospholipid-triglyceride emulsion particles with lipoprotein-depleted plasma, the technique yielded a single peak of highly pure apoA-IV cleanly separated from isoforms of apoA-I and higher molecular weight proteins. A positive linear correlation was observed between apoprotein hydrophobicity and column retention time, thus indicating that the system provided a true reverse phase separation. We conclude that reverse phase high pressure liquid chromatography can separate human apolipoprotein A-IV from isoforms of apoA-I and apoA-II on the basis of differences in mean molecular hydrophobicity. The technique has direct application to the isolation of human apolipoprotein A-IV.

Detection and microsequencing of juvenile hormone-binding proteins of an insect by the use of an iodinated juvenile hormone analog

An [ 125I] iodinated juvenile hormone (JH) analog can be used as a sensitive and highly selective probe for the visualization of high-affinity, (JH)-specific binding proteins from insect hemolymph samples. The proteins can be detected in their native form using a two-dimensional (isoelectric focusing then native gradipore gel) separation of the crude protein mixture containing the 125I-labeled iodinated JH analog. The proteins can be transferred to activated glass fiber paper by electroblotting, and the location of the bound γ-emitter can be found by exposure of the dried gel or the electroblot to X-ray film. The radiolabeled protein spot can be excised from the Coomassie-stained glass fiber paper and subjected directly to gas-phase N-terminal amino acid sequencing. This non-destructive, non-denaturing technique may have wide applicability in identifying and sequencing ligand-specific binding proteins in complex mixtures.

Purification and properties of human placental NADPH-cytochrome P-450 reductase.

Human placental NADPH-cytochrome P-450 reductase (EC 1.6.2.4) was purified to electrophoretic homogeneity in two chromatographic steps with a high retention of bioactivity. After solubilization with 1% sodium cholate in a protective medium containing 20% glycerol, 10 microM 4-androstene-3,17-dione, 1 mM dithiothreitol, and 0.2 mM EDTA, a 35-60% ammonium sulfate precipitate was prepared. The crude protein mixture was then applied to a 2',5'-ADP-Sepharose 4B affinity column, followed by high-performance anion-exchange chromatography (Pharmacia Mono-Q column). Two forms of the reductase were isolated. One was eluted at higher salt concentration and had a relative mass (Mr) of 79 kdaltons (kDa) as estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and high-performance gel permeation chromatography. A smaller size reductase with a Mr of 70 kDa, eluting at lower salt concentration, was also formed by trypsinolysis of the 79-kDa reductase. It must therefore be regarded as a proteolytic artifact. The absolute spectra in the visible region of the two reductases were identical with maxima at 376 and 452 nm, typical of a flavoprotein. They also had the same specific activity of 24.7 +/- 0.7 mumol/min per milligram protein towards cytochrome c. However, only the 79-kDa reductase showed aromatase-reconstitution activity. The homogeneity of these reductases was further confirmed by the appearance of a single peak when subjected to gradient, reversed-phase high-performance liquid chromatography. According to its amino acid composition, the 79-kDa reductase is a highly acidic and hydrophobic protein, composed of 695 residues.

Adsorption chromatography on toyopearl: A single-step alternative to salt fractionation of protein mixtures

Toyopearl media for gel filtration tend to adsorb proteins at high ionic strength, presumably by hydrogen bonding. This is used in the technique proposed here for resolution of crude protein mixtures and initial purification of their components. Proteins can be selectively adsorbed on a column of Toyopearl at high ammonium sulfate concentration and then eluted by decreasing the salt concentration. This single-step procedure can replace the usual salt fractionation of protein mixtures, which is demonstrated with yeast cytochrome oxidase.

22] Isolation and characterization of bovine brain calcineurin: A calmodulin-stimulated protein phosphatase

This chapter focuses on the isolation and characterization of the bovine brain calcineurin. Calcineurin is a heterodimer composed of two subunits, a 19,000 Mr Ca2+-binding subunit, calcineurin B, and a 61,000 Mr subunit, calcineurin A, which interacts with calmodulin. A Ca2+-dependent, calmodulin-stimulated protein phosphatase activity has been found to be associated with calcineurin. The protein can be purified on the basis of its ability to interact with calmodulin after sodium dodecyl sulphate- (SDS)-gel electrophoresis, as well as by its protein phosphatase activity. Upon SDS-polyacrylamide gel electrophoresis, the small subunit of calcineurin, calcineurin B, can be readily identified as a small polypeptide with an apparent Mr between 16,000 and 15,000 that exhibits a characteristic increase in mobility in the presence of Ca2+. Once identified, this subunit can be quantitated by densitometric analysis of slab gels stained with Coomassie Brilliant Blue. The 61,000 Mr subunit, calcineurin A, is more difficult to identify on Coomassie Blue-stained gels of crude protein mixtures because of the presence of many polypeptides of similar molecular weight. However, calcineurin A, the calmodulin-binding subunit of calcineurin, can interact with calmodulin even after SDS-gel electrophoresis and it can be identified and quantitated by a modification of the [125I]calmodulin gel overlay technique.