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1.
Jean A. Boutin Anne-Pascale Ernould Gilles Ferry Annie Genton 《Journal of chromatography. B, Analytical technologies in the biomedical and life sciences》1992,583(2)
A new HPLC method has been developed to assay tyrosine protein kinase activity. Using hydrophilic interaction chromatography, it is possible to resolve the four components of the incubation medium: substrate peptide, [32P]phosphorylated peptide, unreacted [γ-32P]ATP, and 32P-labelled inorganic phosphate. ATP interacts so strongly with the stationary phase material that it can be removed selectively from the incubation medium with solid-phase extraction cartridges packed with the same type of material. The three remaining components of interest can then be resolved by reversed-phase or hydrophilic interaction HPLC. This procedure permits the evaluation of almost every type of peptide as a substrate of tyrosine protein kinase. 相似文献
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Protein chromatography on ion exchange cellulose 总被引:46,自引:0,他引:46
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近年来,乳酸菌细菌素在食品防腐剂和医药领域有着广泛的应用前景,而细菌素的分离纯化是其分子结构及遗传学特性等相关研究的重要基础。离子交换色谱是细菌素分离纯化的主要手段之一。本文阐述了离子交换色谱原理,分析了影响离子交换色谱分离纯化细菌素的各种因素,探讨了细菌素分离纯化中离子交换色谱条件的选择。 相似文献
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Harinarayan C Mueller J Ljunglöf A Fahrner R Van Alstine J van Reis R 《Biotechnology and bioengineering》2006,95(5):775-787
Protein dynamic binding capacities on ion exchange resins are typically expected to decrease with increasing conductivity and decreasing protein charge. There are, however, conditions where capacity increases with increasing conductivity and decreasing protein charge. Capacity measurements on two different commercial ion exchange resins with three different monoclonal antibodies at various pH and conductivities exhibited two domains. In the first domain, the capacity unexpectedly increased with increasing conductivity and decreasing protein charge. The second domain exhibited traditional behavior. A mechanism to explain the first domain is postulated; proteins initially bind to the outer pore regions and electrostatically hinder subsequent protein transport. Such a mechanism is supported by protein capacity and confocal microscopy studies whose results suggest how knowledge of the two types of IEX behavior can be leveraged in optimizing resins and processes. 相似文献
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Plants have attracted interest as hosts for protein expression because of the promise of a large production capacity and a
low production cost. However, recovery costs remain a challenge as illustrated for recovery of recombinant aprotinin, a trypsin
inhibitor, with removal of native corn trypsin inhibitor from transgenic corn (Azzoni et al. in Biotechnol Bioeng 80:268–276,
2002). When expression is targeted to corn grain fractions, dry milling can separate germ and endosperm fractions. Hence,
only the product-containing fraction needs to be extracted, reducing the cost of extraction and the impurity level of the
extract. Selective extraction conditions can reduce impurity levels to the point that low-cost adsorbents can result in relatively
high purity levels. In this work, we attempted to achieve comparable purity with these lower cost methods. We replaced whole
grain extraction and purification of recombinant aprotinin with sequential trypsin affinity and IMAC steps with an alternative
of germ fraction extraction and purification with ion exchange and hydrophobic interaction chromatography (HIC). Using germ
extraction at acidic pH supplemented with heat precipitation to remove additional host proteins resulted in a higher specific
activity feed to the chromatographic steps. The cation exchange step provided 7.6× purification with 76.4% yield and no sodium
dodecyl sulfate–polyacrylamide gel electrophoresis detectable native corn trypsin inhibitor. After the HIC step (2.7× step
purification with 44.0% yield), the final product had a specific activity that was 75.3% of that of the affinity-purified
aprotinin. 相似文献
7.
An ion exchange chromatography process was developed to separate the main protein fractions of bovine blood plasma using a composite material, Q-HyperD resin, and a gel material, DEAE-Sepharose. The experiments were carried out at semipreparative scale. It was necessary to establish analytical methods of electrophoresis and HPLC to identify the fractionated proteins. Results show that these materials are able to adequately fractionate different protein groups from the raw blood plasma. This method may be used to avoid chemical fractionation using agents such as ethanol or PEG and, thus, decrease protein denaturation of the different fractions to be used for research or pharmaceutical purposes. The Q-HyperD resin presents a better retention capacity for plasma protein than DEAE-Sepharose under the experimental conditions employed. 相似文献
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We have developed an improved method for purification of hormone-sensitive lipase from adipose tissue. The method employs two preparative high-performance ion-exchange chromatography steps on Mono Q and Mono S after detergent solubilization and partial fractionation of the enzyme by gradient sievorptive chromatography on QAE-Sephadex. About 0.2 mg of greater than 70% pure enzyme is prepared at 10% yield within 6-7 days from adipose tissue of 500 rats. This protocol is a major improvement over the previously established procedure in terms of accessibility, rapidity, enzyme purity, and yield. 相似文献
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Kisay Lee 《Biotechnology and Bioprocess Engineering》1997,2(2):117-121
Glutamic acid can be crystallized inside cation exchange column when displacer NaOH concentration is high enough to concentrate
displaced glutamic acid beyond its solubility limit. Resulting crystal layer of glutamic acid was moved with liquid phase
through the column, and thus could be eluted from the column and recovered in fraction collector. For the purpose of enhancing
crystal recovery, effects of operating parameters on the crystal formation were investigated. The increase in the degree of
crosslinking of resin favored crystal recovery because of its low degree of swelling. Higher concentration of displacer NaOH
was advantageous. If NaOH concentration is too high, however, crystal recovery was lowered due to the solubility-enhancing
effects of high pH and ionic strength. The decrease of mobile phase flow rate enhanced crystal recovery because enough time
to attain local equilibrium could be provided, but film diffusion would control the overall crystal formation with extremely
low flow rate. Lower temperature reduced solubility of glutamic acid and thus favored crystal formation unless the rate of
ion exchange was severely reduced. The ion exchange operated by displacement mode coupled with crystallization was advantageous
in reducing the burden of further purification steps and in preventing purity-loss resulted from overlapping between adjacent
bands. 相似文献
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IgM and IgG immunoglobulins of human sera were separated by stepwise column chromatography in QAE-Sephadex A-25 ion exchanger gel bed. The procedure resulted within 30 min in a fraction suitable for direct titration of rubella-specific IgM antibodies by haemagglutination inhibition test. The method proved to be a useful diagnostic tool for primary rubella. Serum samples of 13 individuals with previously acquired immunity, 152 patients with a recent rubella-like illness, and 194 pregnant women exposed to rubella infection were tested for the presence of rubella-specific IgM antibodies. Sera of individuals with previous immunity proved to be negative for specific IgM antibodies. Specific IgM titre was demonstrated in the blood of all the 25 patients with significant titre-rise tested because of rubella-like illness, and also in the sera of additional 8 patients whose serum samples were taken too late for demonstration of a rise in titre. Significant titre-rises were found in 5 women exposed to rubella infection, but only two of them exhibited rubella-specific IgM antibodies. The absence of specific IgM antibodies refers presumably to subclinical reinfection in the other three cases. 相似文献
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Denaturation of target protein by various separation and purification steps contributes significant part to the total product loss in bioseparation. The conformational change and accompanying loss of activity of tumor necrosis factor- during ion exchange chromatography was reversible and was decreased by adding polyethylene glycol 200 at 2 to 5% (v/v) to the eluting solution. 相似文献
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Two different approaches of matrix assisted refolding have been evaluated and compared to conventional refolding by dilution. Bovine alpha-lactalbumin was used for the studies as model protein. It was adsorbed under denaturing conditions on an ion exchange matrix and refolding was completed on the column prior to elution or, depending on the buffer system, in the eluate. Agarose based chromatography matrices showed high capacities for the denatured alpha-lactalbumin. A positive effect on the yield of refolded protein by the matrix could be observed for Fractogel EMD DEAE and a negative for Toyopearl DEAE 650M, DEAE Sepharose FF and Q Sepharose FF. In the case of Fractogel EMD DEAE the ion exchange surface might act as a folding helper. This property may be caused by the grafted polymers. For Source 30Q only a marginal negative influence on the refolding kinetics was observed, thus the ion exchanger is only a mean for removal of chaotropic agents. Refolding on the column is characterized by a low yield but high productivity due to significant reduction of refolding time. 相似文献
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Methods for the isolation of glycolytic intermediated by column chromatography with ion exchange resins 总被引:12,自引:0,他引:12
BARTLETT GR 《The Journal of biological chemistry》1959,234(3):459-465
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Large scale chromatographic separation of proteins can be carried out more rapidly on rigid adsorbents than on soft gel media. The kinetics of adsorption of bovine serum albumin (BSA) have been studied on rigid adsorbents based on a wide-pore, hydrophilically-coated silica gel matrix in a packed bed (chromatographic column). Process parameters have been varied comprehensively. The effects of surface chemistry (weak anion exchanger and hydrophobic interaction), particle size and liquid flow velocity have been studied on both the adsorption and desorption processes. The relative influences of the adsorption kinetics and equilibrium isotherm on the shape of the breakthrough curve are found to vary with the process parameters in an interpretable and therefore, predictable manner. Pore diffusion resistance is dominant over the external liquid film resistance in controlling the adsorption kinetics, with Biot numbers in the range 170-2600. A two-step model based on these two resistances simulates the breakthrough curves with only limited quantitative accuracy, but gives good predictions of the effect of changes in process parameters. 相似文献
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A heat-stable enterotoxin produced by human enterotoxigenic Escherichia coli has been purified to homogeneity by hydrophobic interaction, molecular-sieve and high performance liquid chromatography with a recovery of 48%. The toxin is composed of 10 different amino acids with a total of 18 amino acid residues, one-third of which are half-cystine. Purified enterotoxin contains no carbohydrate and is biologically active in the suckling mouse test in 2.1-ng quantities. The molecule was heat-stable (80 degrees C, 20 min.) at pH 7 and pH 2 but lost biological activity at pH 12. Biological activity was lost when treated with the reducing agent dithiothreitol, suggesting that the presence of disulfide bridges is required for biological activity. 相似文献
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