Purification of Cortactin Using Anion Exchange Chromatography

Cortactin is an actin-binding protein that has been shown to be involved in cellular migration and metastases in cancer. Bacterially expressed and purified cortactin protein is often used in in vitro assays to examine cortactin’s role in promoting cell migration via actin remodeling. Cortactin has a theoretical molecular weight of 60 kDa; however, using SDS-PAGE analysis, the protein runs as two bands of molecular weights 80 kDa and 85 kDa, suggesting that cortactin has an unusual protein folding pattern. Our current lack of understanding of cortactin structure limits our ability to determine the role of cortactin in facilitating motility phenotypes. To elucidate the forms of cortactin produced from bacterially expressed and purified cortactin protein, we used a two-step purification system including affinity purification and anion exchange chromatography. After analysis with non-denaturing polyacrylamide gel electrophoresis, we found cortactin protein from different anion chromatography elution fractions did not separate to similar locations on the gel across all fractions. We hypothesize that the variations in the bands are a result of different folding patterns of cortactin protein in what was once thought of as a homogenous protein pool. When testing the role of cortactin in mediating cell migration, these folding differences may have significant effects on the results of functional assays such as actin polymerization or sedimentation

Selectivity variations in anion chromatography

Various organic acids are tested for use as eluents in single-column ion chromatography. Factors affecting their selectivity, sensitivity, and eluting strength are discussed. Coated anion exchange resins suitable for anion chromatography are also prepared. Their convenience, flexibility, and practical use as anion exchangers are demonstrated;Theories on ion exchange selectivity with high capacity ion exchange resins are reviewed. Interactions emphasized by each theory are pointed out as well as possible problems that could arise when applying these theories to explain selectivity variations found in anion chromatography. Previous studies in anion chroma- tography which exhibited selectivity variations are also reviewed and explanations for the variations are given. Parameters of the chromatographic system that could affect the selectivity of the sys- tem for various anions are investigated. Then, selectivity variations are explained in terms of interactions between components of the mobile phase and the stationary phase;Derivatization techniques necessary for species that are difficult or impossible to detect directly by anion chromatography are investigated. Methods are described for derivatizing cyanide and;aldehydes in aqueous solution and then determining them by anion chromatography; (\u271)DOE Report IS-T-1169. This work was performed under Contract W-7405-eng-82 with the Department of Energy

A multi-analytical approach to better assess the keratan sulfate contamination in animal origin chondroitin sulfate

Abstract Chondroitin sulfate is a glycosaminoglycan widely used as active principle of anti-osteoarthritis drugs and nutraceuticals, manufactured by extraction from animal cartilaginous tissues. During the manufacturing procedures, another glycosaminoglycan, the keratan sulfate, might be contemporarily withdrawn, thus eventually constituting a contaminant difficult to be determined because of its structural similarity. Considering the strict regulatory rules on the pureness of pharmaceutical grade chondrotin sulfate there is an urgent need and interest to determine the residual keratan sulfate with specific, sensitive and reliable methods. To pursue this aim, in this paper, for the first time, we set up a multi-analytical and preparative approach based on: i) a newly developed method by high performance anion-exchange chromatography with pulsed amperometric detection, ii) gas chromatography-mass spectrometry analyses, iii) size exclusion chromatography analyses coupled with triple detector array module and on iv) strong anion exchange chromatography separation. Varied KS percentages, in the range from 0.1 to 19.0% (w/w), were determined in seven pharmacopeia and commercial standards and nine commercial samples of different animal origin and manufacturers. Strong anion exchange chromatography profiles of the samples showed three or four different peaks. These peaks analyzed by high performance anion-exchange with pulsed amperometric detection and size exclusion chromatography with triple detector array, ion chromatography and by mono- or two-dimensional nuclear magnetic resonance revealed a heterogeneous composition of both glycosaminoglycans in terms of sulfation grade and molecular weight. High molecular weight species (>100 KDa) were also present in the samples that counted for chains still partially linked to a proteoglycan core

DESIGN OF ADVANCED ION-EXCHANGE MEMBRANES AND THEIR PERFORMANCE ASSESSMENT FOR DOWNSTREAM CHROMATOGRAPHIC BIOSEPARATIONS

This doctoral research focuses on the design, development and characterization of advanced ion-exchange membranes and their performance evaluation as process chromatography media for downstream bioseparations. Chromatography is a widely used unit operation in the biopharmaceutical industry for the downstream purification of protein therapeutics. The rapid developments in biotechnology and the pharmaceutical potential of biomolecules have increased the worldwide demand for protein therapeutics dramatically. Considering that 50−90% of the total cost of bioprocesses is due to the downstream recovery and purification, high-productivity and high-resolution separation techniques that will enable cost-effective production are essential to the biopharmaceutical industry. In recent years, membrane chromatography has been promoted as a promising alternative to more conventional packed-bed resin chromatography. Although the potential for membrane chromatography is great, the historically lower binding capacity of membranes compared to resin media has limited its broad implementation. Therefore, primary objectives of this dissertation were to prepare advanced weak and strong anion-exchange membranes with ultrahigh and completely reversible protein binding capacities and to demonstrate the high-throughput and high resolution that these membranes enable in the separation of a target protein from a complex media (cell lysate). The research presented here pertains to the use of atom transfer radical polymerization (ATRP) to prepare surface-modified weak and strong anion-exchange membranes for chromatographic bioseparations. Surface-initiated atom transfer radical polymerization (ATRP) was used to graft poly(2-dimethylaminoethyl methacrylate), (poly(DMAEMA)), and poly([2-(methacryloyloxy)ethyl]trimethylammonium chloride), (poly(MAETMAC)), nanolayers from the internal pore surfaces of commercial regenerated microporous membranes. Characterization of physicochemical and performance properties of newly designed, surface-modified membranes was performed using various analytical techniques. The central theme of my research was to investigate how polymer architecture influences the separation performance properties of surface-modified ion-exchange membranes. In one study, the grafting density and average molecular weight of polymer chains grown from the membrane pore surfaces were varied independently and optimized to prepare weak anion-exchange membranes with ultra-high and completely reversible dynamic binding capacity. The effects of polymer grafting density, average molar mass of polymer and linear flow velocity on the dynamic binding capacity were studied. This study yielded weak anion-exchange membranes with very high volumetric protein binding capacities (static binding capacity∼140 mg BSA/mL and dynamic capacity ∼130 mg/mL) at high linear flow velocities (\u3e350 cm/h) and relatively low transmembrane pressure drop (\u3c3 bar). In a second study, a systematic evaluation was performed on the role of polymer molecular architecture on the separation performance of strong anion-exchange membranes. Anion-exchange membranes with different polymer chain densities were prepared using surface-initiated ATRP. The effect of polymer chain density, and, thus the, degree of polymer grafting, on the mass transfer resistances and accessibility of large biopolymers (IgG and DNA) was studied. From this detailed study, I have prepared a unique protocol to design strong Q-type anion-exchange membranes with unusually high volumetric protein binding capacities (dynamic binding capacity ∼140 mg IgG/mL and ∼27 mg DNA/mL) at high linear flow velocities (\u3e190 cm/h) and relatively low transmembrane pressure drop (\u3c3.5 bar). Overall, findings from my Doctor of Philosophy (PhD) studies strengthen the argument that membrane chromatography can be a higher capacity and higher throughput process than resin chromatography. Finally, I evaluated the protein separation performance of my newly designed anion-exchange membrane adsorber and compared it to a commercial membrane adsorber and resin column. One aspect of this study was to compare the protein separation performance of membrane chromatography with resin column chromatography. Anion-exchange chromatography was used under salt-gradient and pH-gradient elution to separate anthrax protective antigen protein from periplasmic Escherichia coli lysate. Overall, this part of the work demonstrates that membrane chromatography is a high-capacity, high-throughput, high-resolution separation technique, and that resolution in membrane chromatography can be higher than resin column chromatography under preparative conditions and at much higher (15 times higher than widely used resin column) volumetric throughput

Purification and analytical characterization of an anti- CD4 monoclonal antibody for human therapy

A purification process for the monclonal anti-CD4 antibody MAX.16H5 was developed on an analytical scale using (NH&SO, precipitation, anion-exchange chromatography on MonoQ or Q-Sepharose, hydrophobic interaction chromatography on phenyl- Sepharose and gel filtration chromatography on Superdex 200. The purification schedule was scaled up and gram amounts of MAX.16H5 were produced on corresponding BioPilot columns. Studies of the identity, purity and possible contamination by a broad range of methods showed that the product was highly purified and free from contaminants such as mouse DNA, viruses, pyrogens and irritants. Overall, the analytical data confirm that the monoclonal antibody MAX.16H5 prepared by this protocol is suitable for human therapy

New stationary phases for efficient separations and selectivity studies in anion chromatography

Spacer arm anion-exchange resins for single-column ion chromatography (SCIC) were prepared by a two-step procedure consisting of a bromoalkylation of Rohm & Haas XAD-1 resin under mild conditions, followed by amination with trimethylamine. The five resins prepared are all of a similar, low capacity and contain a one-, two-, three-, four- or six-carbon spacer arm linkage between the resin surface and the quaternary ammonium exchange group. The selectivity of these resins for mono- and divalent anions is evaluated and explained using classical ion-exchange theory. Separations are presented to demonstrate resin selectivity and to show the usefulness of these resins for practical ion chromatography;Trimethylammonium, tributylammonium and tributylphosphonium anion-exchange resins were synthesized and used for SCIC. The three resins were prepared using a 7 to 12 [mu]m polystyrene-divinylbenzene resin and are all of a similar, low capacity. The selectivity of these resins for mono- and divalent anions is examined and related to classical theory. It is shown that the unique selectivity obtained with the quaternary phosphonium resin is of practical value in the separation of anions;A new stationary phase for anion chromatography was prepared by a simple method of coating quaternized latex particles onto the surface of unsulfonated polymeric resins. A nonporous polystyrene resin of very uniform particle size is described that serves as an excellent substrate for the coated resins. These latex-coated resins are used to obtain highly efficient anion separations

Electrophoretic Heterogeneity of Nicotinamide Adenine Dinucleotide Phosphate-Dependent 5-Ketogluconate Reductase of Gluconobacter Suboxydans

NADP+-dependent 5-ketogluconate reductase (D-Gluconate:NAD(P)+ oxidoreductase E.C. 1.1.1.69) isozymes were detected in cell-free extracts of Gluconobacter suboxydans (American Type Cultures Collection, Bethesda, Maryland, strain 621). Cell-free enzyme extracts were prepared from cells grown in media which contained glucose, gluconate, glycerol, sorbitol, or mannitol as the sole carbon source in 2% concentration. The extracts were fractionated using analytical polyacrylamide disc-gel electrophoresis. Gels were stained specifically for 5-ketogluconate reductase. Three isozymes were present in each extract from cells grown on the five different carbon sources. Gels stained in a reaction mixture which contained glycerol exhibited two of the isozymes. An attempt was made to separate the three isozymes using anion-exchange chromatography. A stepwise anion-exchange procedure on DEAE Sephadex resulted in the separation of one of the isozymes from the other two and indicated that this isozyme might actually be two isozymes with the same electrophoretic mobility. Results fro linear gradient anion-exchange chromatography disproved this possibility. A comparison of results obtained from tracticelation by linear gradient anion-exchange of extracts which had been previously fractionated on Sephadex Gel-100 gel and those which had not, indicated that gel filtration did not improve separation of the isozymes

Separación de proteínas de suero de leche por cromatografía líquida

This paper describes and compares three chromatographic methods for the analysis and quantification of most abundant proteins in cheese whey, α-lactalbumin and β-lactoglobulin. The methods were: Reverse-phase high performance liquid chromatography, anion Exchange chromatography and size-exclusion chromatography. The reverse- phase liquid chromatography led to a better separation of whey proteins than size-exclusion chromatography and anion exchange chromatography, this method offered an excellent separation for whey proteins and presented a short time of analysis (33 min).Este artículo describe y compara tres métodos cromatográficos para el análisis y la cuantificación de las proteínas más abundantes en el suero de queso, α-lactoglobulina y β-lactoalbúmina. Los métodos fueron los siguientes: cromatografía líquida de alta eficacia en fase reversa, cromatografía de intercambio aniónico y cromatografía de exclusión molecular. La cromatografía líquida en fase reversa condujo a una mejor separación de las proteínas de suero de leche que la cromatografía de exclusión molecular y la cromatografía de intercambio aniónico, este método ofrece una excelente separación de las proteínas de suero de leche, y presentó un breve tiempo de análisis (33 min)