Ni2+-based immobilized metal ion affinity chromatography of lactose operon repressor protein from Escherichia coli

A two-step chromatographic sequence is described for the purification of native lactose operon repressor protein from Escherichia coli cells. The first step involves Ni2+-based immobilized metal ion affinity chromatography of the soluble cytoplasmic extract. This method provides superior speed, resolution and yield than the established phosphocellulose cation-exchange chromatographic procedure. Anion-exchange chromatography is used for further purification to >95% purity. The identity and purity of the lactose repressor protein were demonstrated using sodium dodecylsulphate polyacrylamide electrophoresis, crystallization, tryptic finger-printing mass spectrometry, and inducer binding assays. The purified lac repressor exhibited inducer sensitivity for operator DNA binding and undergoes a conformational change upon inducer binding. By all these extensive biochemical criteria, the purified protein behaves exactly as that described for the Escherichia coli lactose operon repressor.

Polystyrene-type resin used for peptide synthesis: application for anion-exchange and affinity chromatography

This paper deals with an unusual application for a copolymer of styrene-1 % divinylbenzene bearing high amount of aminomethyl groups for anion-exchange and affinity chromatography. The so-called aminomethyl resin (AMR), to date only employed for peptide synthesis, swelled appreciably in water and was used successfully to purify negatively charged peptides. By correlating swelling degree of beads with pH of the media, it was possible to estimate that the AMR amino group pK(a) is approximately 5.5. In addition, the synthesized acetyl-(NANP)(3)-AMR succeeded in the affinity interaction with large antibody molecules related to malaria transmission and raised previously against this dodecapeptide sequence. (C) 2004 Elsevier B.V. All rights reserved.

Use of commercial anion-exchange resins as solid support for peptide synthesis and affinity chromatography

This report demonstrates that due to the presence of residual reactive sites in their matrices, classical diethylaminoethyl-attaching commercial anion-exchanger resins such as DEAE-MacroPrep and DEAE-Sephadex A50 supports can be used for peptide synthesis. Moreover, due to the high stability of the peptide-resin bond in the final cleavage treatments, desired peptidyl-resins free of side-chain protecting groups, which enables them to be further used as solid support for affinity chromatography, can be obtained. To demonstrate this potentiality, a fragment corresponding to the antigenic and immunodominant epitope of sporozoites of the Plasmodium falciparum malaria parasite was synthesized in these traditional resins and antibody molecules generated against the peptide sequence were successfully retained in these peptidyl supports. Due to the maintenance of their original anion-exchange capacities, the present findings open the unique possibility of applying, simultaneously, dual anion-exchange and affinity procedures for purification of a variety of macromolecules. (C) 2003 Elsevier B.V. (USA). All rights reserved.

Purification and partial characterization of cathepsin D from porcine (Sus scrofa) liver using affinity chromatography

Cathepsin D, a lysosomal aspartic protease, has been purified from porcine liver using a combination of pepstatin-A agarose and Affi-Gel Blue affinity chromatography, followed by size-exclusion chromatography. The purified protein consists of two polypeptide chains of 15 and 30 kDa, and has an isoelectric point of 6.8. Porcine liver cathepsin D has maximum activity at pH 2.5-3.0 as determined by its activity against hemoglobin, with a K-cat of 14.3 s(-1) and a k(cat)/K-M of 2.70 x 10(6) s(-1) M-1 as determined by the hydrolysis of a fluorogenic peptide substrate.

Use of chemically modified silica with beta-diketoamine groups for separation of alpha-lactoalbumin from bovine milk whey by affinity chromatography

Silica gel surface was chemically modified with beta-diketoamine groups by reacting the silanol from the silica surface with 3-aminopropyl-triethoxysilane and 3-bromopentanedione, With this material, copper ions were adsorbed from aqueous solutions, the chemical analysis of the silica-gel-immobilized acetylacetone provided a quantity of 0.67 mmol g(-1) of organic groups attached to the support and 0.63 mmol g(-1) of copper, This material was used as a stationary phase in IMAC (immobilized metal affinity chromatography), to separate alpha-lactoalbumin from bovine milk whey, the results showed an efficient separation in the chromatographic column, the possibility of reutilization of the stationary phase was also investigated. (C) 1997 Academic Press

Comparison of affinity chromatography supports for separation of protein

Chromatography is the most widely used technique for high-resolution separation and analysis of proteins. This technique is very useful for the purification of delicate compounds, e.g. pharmaceuticals, because it is usually performed at milder conditions than separation processes typically used by chemical industry. This thesis focuses on affinity chromatography. Chromatographic processes are traditionally performed using columns packed with porous resin. However, these supports have several limitations, including the dependence on intra-particle diffusion, a slow mass transfer mechanism, for the transport of solute molecules to the binding sites within the pores and high pressure drop through the packed bed. These limitations can be overcome by using chromatographic supports like membranes or monoliths. Dye-ligands are considered important alternatives to natural ligands. Several reactive dyes, particularly Cibacron Blue F3GA, are used as affinity ligand for protein purification. Cibacron Blue F3GA is a triazine dye that interacts specifically and reversibly with albumin. The aim of this study is to prepare dye-affinity membranes and monoliths for efficient removal of albumin and to compare the three different affinity supports: membranes and monoliths and a commercial column HiTrapTM Blue HP, produced by GE Healthcare. A comparison among the three supports was performed in terms of binding capacity at saturation (DBC100%) and dynamic binding capacity at 10% breakthrough (DBC10%) using solutions of pure BSA. The results obtained show that the CB-RC membranes and CB-Epoxy monoliths can be compared to commercial support, column HiTrapTM Blue HP, for the separation of albumin. These results encourage a further characterization of the new supports examined.

Determination of binding constants by affinity chromatography

This review summarizes developments in the use of affinity chromatography to characterize biospecific interactions in terms of reaction stoichiometry and equilibrium constant. In that regard, the biospecificity incorporated into the design of the experiment ensures applicability of the method regardless of the sizes of the reacting solutes. By the adoption of different experimental strategies (column chromatography, simple partition equilibrium, solid-phase immunoassay and biosensor technology protocols) quantitatiative affinity chromatography can be used to characterize interactions governed by an extremely broad range of binding affinities. In addition, the link between ligand-binding studies and quantitative affinity chromatography is illustrated by means of partition equilibrium studies of glycolytic enzyme interactions with muscle myofibrils. an exercise which emphasizes that the same theoretical expressions apply to naturally occurring examples of affinity chromatography in the cellular environment. (C) 2004 Elsevier B.V. All rights reserved.

Affinity membrane chromatography for the analysis and purification of proteins

Affinity chromatography is unique among separation methods as it is the only technique that permits the purification of proteins based on biological functions rather than individual physical or chemical properties. The high specificity of affinity chromatography is due to the strong interaction between the ligand and the proteins of interest. Membrane separation allows the processing of a large amount of sample in a relatively short time owing to its structure, which provides a system with rapid reaction kinetics. The integration of membrane and affinity chromatography provides a number of advantages over traditional affinity chromatography with porous-bead packed columns, especially with regard to time and recovery of activity. This review gives detailed descriptions of materials used as membrane substrates, preparation of basic membranes, coupling of affinity ligands to membrane supports, and categories of affinity membrane cartridges. It also summarizes the applications of cellulose/glycidyl methacrylate composite membranes for proteins separation developed in our laboratory. (C) 2001 Elsevier Science B.V. All rights reserved.

Identification of salivary N-glycoproteins and measurement of glycosylation site occupancy by boronate glycoprotein enrichment and liquid chromatography/electrospray ionization tandem mass spectrometry

RATIONALE Diseases including cancer and congenital disorders of glycosylation have been associated with changes in the site-specific extent of protein glycosylation. Saliva can be non-invasively sampled and is rich in glycoproteins, giving it the potential to be a useful biofluid for the discovery and detection of disease biomarkers associated with changes in glycosylation. METHODS Saliva was collected from healthy individuals and glycoproteins were enriched using phenylboronic acid based glycoprotein enrichment resin. Proteins were deglycosylated with peptide-N-glycosidase F and digested with AspN or trypsin. Desalted peptides and deglycosylated peptides were separated by reversed-phase liquid chromatography and detected with on-line electrospray ionization quadrupole-time-of-flight mass spectrometry using a 5600 TripleTof instrument. Site-specific glycosylation occupancy was semi-quantitatively determined from the abundance of deglycosylated and nonglycosylated versions of each given peptide. RESULTS Glycoprotein enrichment identified 67 independent glycosylation sites from 24 unique proteins, a 3.9-fold increase in the number of glycosylation sites identified. Enrichment of glycoproteins rather than glycopeptides allowed detection of both deglycosylated and nonglycosylated versions of each peptide, and thereby robust measurement of site-specific occupancy at 21 asparagines. Healthy individuals showed limited biological variability in occupancy, with partially modified sites having characteristics consistent with inefficient glycosylation by oligosaccharyltransferase. Inclusion of negative controls without enzymatic deglycosylation controlled for spontaneous chemical deamidation, and identified asparagines previously incorrectly annotated as glycosylated. CONCLUSIONS We developed a sample preparation and mass spectrometry detection strategy for rapid and efficient measurement of site-specific glycosylation occupancy on diverse salivary glycoproteins suitable for biomarker discovery and detection of changes in glycosylation occupancy in human disease.

Binding properties of peptidic affinity ligands for plasmid DNA capture and detection

Peptides constructed from α-helical subunits of the Lac repressor protein (LacI) were designed then tailored to achieve particular binding kinetics and dissociation constants for plasmid DNA purification and detection. Surface plasmon resonance was employed for quantification and characterization of the binding of double stranded Escherichia coli plasmid DNA (pUC19) via the lac operon (lacO) to "biomimics" of the DNA binding domain of LacI. Equilibrium dissociation constants (K D), association (k a), and dissociation rates (k d) for the interaction between a suite of peptide sequences and pUC19 were determined. K D values measured for the binding of pUC19 to the 47mer, 27mer, 16mer, and 14mer peptides were 8.8 ± 1.3 × 10 -10 M, 7.2 ± 0.6 × 10 -10 M, 4.5 ± 0.5 × 10 -8 M, and 6.2 ± 0.9 × 10 -6 M, respectively. These findings show that affinity peptides, composed of subunits from a naturally occurring operon-repressor interaction, can be designed to achieve binding characteristics suitable for affinity chromatography and biosensor devices.

Binding mechanism of affinity ligands for purification of plasmid DNA

Plasmid DNA for therapeutic and vaccination purposes must be highly purified. The high selectivity of affinity chromatography makes it ideal for the isolation of pDNA from complex biological feed stocks. Affinity chromatography makes use of the biological function and/or individual chemical structure of the interacting molecules. However, the success of any affinity purification protocol is dependent on the availability of suitable ligands. In this study, surface plasmon resonance (SPR) based Biacore system has been employed for the detection and quantification of the binding between lac operon (lacO) sequence contained in a pDNA and synthetic peptides based on the DNA-binding domain of the lac repressor protein, lad. The equilibrium dissociation constant (K D) and association and dissociation rate constants (ka, kd) for the interaction between plasmid DNA and designed peptides were determined.

Affinity purification of plasmid DNA directly from crude bacterial cell lysates

We have shown previously that a sequence-specific DNA-binding protein based on the Lac repressor protein can isolate pre-purified DNA efficiently from simple buffer solution but our attempts to purify plasmids directly from crude starting materials were disappointing with unpractically low DNA yields. We have optimized tbe procedure and present a simple affinity methodology whereby plasmid DNA is purified directly by mixing two crude cell lysates, one cell lysate containing the plasmid and the other the protein affinity ligand, without the need for treatment by RNaseA. After IMAC chromatography, high purity supercoiled DNA is recovered in good yields of 100-150 μg plasmid per 200 mL shake flask culture. Moreover, the resulting DNA is free from linear or open-circular plasmid DNA, genomic DNA, RNA, and protein, to the limits of our detection. Furthermore, we show that lyophilized affinity ligand can be stored at room temperature and re-hydrated for use when required.

LacO-LacI interaction in affinity adsorption of plasmid DNA

Current approaches for purifying plasmids from bacterial production systems exploit the physiochemical properties of nucleic acids in non-specific capture systems. In this study, an affinity system for plasmid DNA (pDNA) purification has been developed utilizing the interaction between the lac operon (lacO) sequence contained in the pDNA and a 64mer synthetic peptide representing the DNA-binding domain of the lac repressor protein, LacI. Two plasmids were evaluated, the native pUC19 and pUC19 with dual lacO3/lacOs operators (pUC19lacO3/lacOs), where the lacOs operator is perfectly symmetrical. The DNA-protein affinity interaction was evaluated by surface plasmon resonance using a Biacore system. The affinity capture of DNA in a chromatography system was evaluated using LacI peptide that had been immobilized to Streamline™ adsorbent. The KD-values for double stranded DNA (dsDNA) fragments containing lacO1 and lacO3 and lacOs and lacO3 were 5.7 ± 0.3 × 10 -11 M and 4.1 ± 0.2 × 10-11 M respectively, which compare favorably with literature reports of 5 × 10-10 - 1 × 10-9 M for native laCO1 and 1-1.2 × 10-10 M for lacO1 in a saline buffer. Densitometric analysis of the gel bands from the affinity chromatography run clearly showed a significant preference for capture of the supercoiled fraction from the feed pDNA sample. The results indicate the feasibility of the affinity approach for pDNA capture and purification using native protein-DNA interaction.

Affinity adsorption of plasmid DNA

The development of a protein-mediated dual functional affinity adsorption of plasmid DNA is described in this work. The affinity ligand for the plasmid DNA comprises a fusion protein with glutathione-S-transferase (GST) as the fusion partner with a zinc finger protein. The protein ligand is first bound to the adsorbent by affinity interaction between the GST moeity and gluthathione that is covalently immobilized to the base matrix. The plasmid binding is then enabled via the zinc finger protein and a specific nucleotide sequence inserted into the DNA. At lower loadings, the binding of the DNA onto the Fractogel, Sepharose, and Streamline matrices was 0.0078 ± 0.0013, 0.0095 ± 0.0016, and 0.0080 ± 0.0006 mg, respectively, to 50 μL of adsorbent. At a higher DNA challenge, the corresponding amounts were 0.0179 ± 0.0043, 0.0219 ± 0.0035, and 0.0190 ± 0.0041 mg, respectively. The relatively constant amounts bound to the three adsorbents indicated that the large DNA molecule was unable to utilize the available zinc finger sites that were located in the internal pores and binding was largely a surface adsorption phenomenon. Utilization of the zinc finger binding sites was shown to be highest for the Fractogel adsorbent. The adsorbed material was eluted with reduced glutathione, and the eluted efficiency for the DNA was between 23% and 27%. The protein elution profile appeared to match the adsorption profiles with significantly higher recoveries of bound GST-zinc finger protein.