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Biomedical subjects

B Mattiasson

Publications and source records attributed to B Mattiasson.

At least 73 records · Page 4Linked to original sources
PubMed1999

'Smart' polymers and what they could do in biotechnology and medicine.

Stimulus-responsive or 'smart' polymers undergo strong conformational changes when only small changes in the environment (e. g. pH, temperature, ionic strength) occur. These changes result in phase separation from aqueous solution or order-of-magnitude changes in hydrogel size. Smart polymers are used in bioseparation and drug delivery, for the development of new biocatalysts, as biomimetic actuators, and as surfaces with switchable hydrophobic-hydrophilic properties.

Biotechnology↗
PubMed1999

A microbial biosensor based on Yarrowia lipolytica for the off-line determination of middle-chain alkanes.

A microbial biosensor based on immobilised psychrotrophic yeast Yarrowia lipolytica integrated to FIA for the determination of middle chain alkanes was developed. The system responded very well to middle chain alkanes even at low operational temperatures down to +5 degrees C. The maximum sensitivity was obtained at 15 degrees C. A linear relationship was observed between the sensor response and dodecane concentration up to 100 microM.

Alkanes↗
PubMed1999

Purification of Lac repressor protein using polymer displacement and immobilization of the protein.

Lac repressor protein was purified from E. coli BMH8117 harboring plasmid pWB1000 and E. coli K12BMH 71-18 strains. Displacement of the protein with poly(ethyleneimine) (PEI) from phosphocellulose cation exchange column was shown to be an effective elution strategy. It resulted in better recoveries and sharper elution profiles than traditional salt elution without effecting the purity of the protein. The elution is assumed to proceed via displacement of bound protein by PEI when the polymer binds to the ion exchanger. The minor impurities in the protein solution were finally removed by chromatography on immobilized metal affinity column. The repressor protein undergoes distinct conformational changes upon addition of specific inducer isopropyl-beta-D-thiogalactoside (IPTG), which is evidenced by changes in ultraviolet absorption spectrum. The protein was immobilized covalently to the Sepharose matrix. The intact biological activity of the protein after immobilization was shown by binding of genomic DNA and lac operator plasmid DNA from E. coli to the immobilized lac repressor.

Bacterial Proteins↗
PubMed1999

Affinity isolation of a cold-adapted enzyme: lactate dehydrogenase from Bacillus psychrosaccharolyticus.

A simple, economical and rapid affinity chromatography procedure with dyes as the ligand has been described for the one-step purification of a cold-adapted lactate dehydrogenase. Non-specific elution of Procion blue H-ERD-modified Sepharose yielded homogeneous preparations of lactate dehydrogenase both in column based procedures and in batch wise operations. Low operational temperatures resulted in the enhanced binding of the enzyme to the blue dye. The dissociation constants of the enzyme-dye complexes were 7.2 +/- 0.2 microM and 11.2 +/- 0.2 microM at 5 degrees C and 20 degrees C respectively.

Bacillus↗
PubMed1999

On-line monitoring of glucose and/or lactate in a fermentation process using an expanded micro-bed flow injection analyser.

A novel flow injection biosensor system for monitoring fermentation processes has been developed using an expanded micro bed as the enzyme reactor. An expanded bed reactor is capable of handling a mobile phase containing suspended matter like cells and cell debris. Thus, while the analyte is free to interact with the adsorbent, the suspended particulate matter passes through unhindered. With the use of a scaled down expanded bed in the flow injection analysis (FIA) system, it was possible to analyse samples directly from a fermentor without the pretreatment otherwise required to extract the analyte or remove the suspended cells. This technique, therefore, provides a means to determine the true concentrations of the metabolites in a fermentor, with more ease than possible with other techniques. Glucose oxidase immobilised on STREAMLINE was used to measure glucose concentration in a suspension of dead yeast cells. There was no interference from the cell particles even at high cell densities such as 15 gm dry weight per litre. The assay time was about 6 min. Accuracy and reproducibility of the system was found to be good. In another scheme, lactate oxidase was covalently coupled to STREAMLINE for expanded bed operation. With the on-line expanded micro bed FIA it was possible to follow the fermentation with Lactobacillus casei.

Bioreactors↗
PubMed1999

Binding assays in heterogeneous media using a flow injection system with an expanded micro-bed adsorption column.

Competitive binding assays have been performed in flow injection systems. To further increase the versatility of the system, and to enable it to deal with samples containing particulate matter, the adsorption step was designed as an expanded bed column. Immunochemical quantification of human serum albumin was chosen as a model system to use for the development of the technology. A competitive ELISA was set up using peroxidase labelled HSA as competing ligand. The introduction of the expanded bed immunosorption column made the system tolerant to samples containing suspended particulate matter. The analytical outcome is very similar to that from the packed bed system even though more time is required for each assay cycle. The capability of the system was tested by addition of increasing amounts of yeast cells. The results clearly indicate that the system is suitable e.g. for process monitoring of fermentations.

Adsorption↗
PubMed1999

Use of a micro-expanded bed containing immobilised lysozyme for cell disruption in flow injection analysis.

A method for cell disruption in Flow Injection Analysis (FIA) systems has been developed. The principle involves on-line cell disruption by means of immobilised lysozyme followed by an ultrasonic treatment. In order to avoid flow problems in the analytical system, the lysozyme was immobilised to Streamline that was used in an expanded bed in the flow system. Samples of suspensions of Micrococcus lysodeikticus were treated and the success of the treatment was evaluated in terms of released protein and as a decrease in the optical density at 450 nm. The new technology offers a powerful tool in flow injection analyses for quantification of intracellular compounds. The concept of integration, i.e. combining cell disruption with handling of cell debris and assay procedure in one continuous flow process facilitates its use and increases the probability of reaching reproducible and reliable results.

Enzymes, Immobilized↗
PubMed1999

Flow injection analysis of intracellular beta-galactosidase in Escherichia coli cultivations, using an on-line system including cell disruption, debris separation and immunochemical quantification.

A continuous integrated process for on-line quantification of intracellular components has been developed. By applying the concept of expanded micro-beds in a flow injection system it was possible to first perform on-line cell disintegration followed by an on-line binding assay for quantification of a reporter protein (beta-galactosidase) from the cell interior. The disintegration process involved the use of an expanded bed with immobilised lysozyme followed by ultrasonic treatment in a flow-through cell. The cell debris does not interfere in the binding assay as it is carried out in an expanded bed. The time for an assay cycle is at present approx. 35 min. This integrated system can be used for quantification of proteins down to at least 10(-7) mol/L.

Calibration↗
PubMed1998

Detection of heavy metal ions at femtomolar levels using protein-based biosensors.

Sensors based on proteins (GST-SmtA and MerR) with distinct binding sites for heavy metal ions were developed and characterized. A capacitive signal transducer was used to measure the conformational change following binding. The proteins were overexpressed in Escherichia coli, purified, and immobilized in different ways to a self-assembled thiol layer on a gold electrode placed as the working electrode in a potentiostatic arrangement in a flow analysis system. The selectivity and the sensitivity of the two protein-based biosensors were measured and compared for copper, cadmium, mercury, and zinc ions. The GST-SmtA electrodes displayed a broader selectivity (sensing all four heavy metal ions) compared with the MerR-based ones, which showed an accentuated selectivity for mercury ions. Metal ions could be detected with both electrode types down to femtomolar concentration. The upper measuring limits, presumably due to near saturation of the proteins' binding sites, were around 10(-10) M. Control electrodes similarly constructed but based on bovine serum albumin or urease did not yield any signals. The electrodes could be regenerated with EDTA and used for more than 2 weeks with about 40% reduction in sensitivity.

Amino Acid Sequence↗
PubMed1998

Affinity precipitation of alpha-amylase inhibitor from wheat meal by metal chelate affinity binding using cu(II)-loaded copolymers of 1-vinylimidazole with N-isopropylacrylamide

A method for purifying alpha-amylase inhibitor from wheat meal based on immobilized metal affinity with a thermosensitive copolymer is developed. The studies represent the thermoprecipitation properties of the copolymers of N-isopropylacrylamide (NIPAM) with iminodiacetic acid (IDA) and 1-vinylimidazole (VI), respectively. The polymer which is obtained by the copolymerization of 1-vinylimidazole and N-isopropylacrylamide, charged with Cu(II), exhibited specific interaction of the metal ions to the protein inhibitor. The precipitation was induced by salt and the recovery of the amylase inhibitor was achieved by dissolving the inhibitor-polymer complex in imidazole buffer and subsequent precipitation of the polymer. A single family of the alpha-amylase inhibitor was recovered from the polymer with 89% yield and about fourfold purification. The SDS-PAGE pattern showed significant purification of the inhibitor. The binding of the inhibitor to the Cu(II)-polymer conjugate depends upon the Cu(II) concentration in the copolymer and also upon the concentration of the protein. The recovered polymer could be reused with reasonable efficiency. Copyright 1998 John Wiley & Sons, Inc.

Journal Article↗
PubMed1998

Conjugates of monoclonal antibodies with polyelectrolyte complexes-- an attempt to make an artificial chaperone.

Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) from rabbit muscle is a tetrameric enzyme. Inactivation of GAPDH at 50 degreesC or in the presence of 4 M urea proceeds via formation of inactive dimers followed by their aggregation. Antibodies (clone 6C5) were selected which bind specifically inactive dimers but not native tetramers. The simplified model of chaperone action when the inactive misfolded forms are removed from the reaction media preventing aggregation was developed using antibodies in combination with polyelectrolyte complexes. The antibodies were coupled covalently to polyanionic component of the complex, poly(methacrylic) acid. The treatment of inactivated GAPDH with this conjugate followed by its precipitation after equimolar addition of polycation, poly-(N-ethyl-4-vinylpyridinium) bromide, resulted in a significant increase in the specific activity of GAPDH. The restoration of specific activity was more complete in the experiments with lower GAPDH concentration and in the samples with lower inactivation degree, conditions where aggregation is less pronounced. Some aggregates are formed at high inactivation degree and high GAPDH concentration and observed as an increase in the solution turbidity. They could be removed by centrifugation. Antibody/polyelectrolyte complex treatment followed by centrifugation to remove insoluble aggregates resulted in nearly complete restoration of enzyme specific activity.

Animals↗
PubMed1998

Affinity precipitation of proteins: design criteria for an efficient polymer.

Affinity precipitation is fast emerging as a successful technique for the purification of proteins which can be introduced at an early stage of downstream processing. The technique applies the use of reversibly soluble-insoluble polymers which have either natural or synthetic origin. Apart from the successful use of some natural polymers, such as chitosan and alginate, the vast application of the technique depends upon the design of efficient synthetic polymers. In this laboratory, N-isopropylacrylamide (NIPAM) copolymers have been developed for metal chelate affinity precipitation of proteins. The copolymers of 1-vinylimidazole (VI) and iminodiacetic acid (IDA) with NIPAM were synthesized. The copolymers were thoroughly characterized with a view to designing an efficient soluble-insoluble polymer for metal chelate affinity precipitation of proteins.

Acrylic Resins↗
PubMed1998

Affinity precipitation of concanavalin A--studies of some underlying mechanisms using dynamic laser light scattering.

The final outcome of an affinity precipitation process will depend upon the efficiency of each individual stage involved: the formation of initial affinity complexes, the build-up of a precipitate and the elution of the target protein. Investigations on the first stage were done in this study utilizing a model system. The target protein was the lectin concanavalin A (Con A). Eudragit S-100, a reversibly soluble/insoluble polymer consisting of methyl methacrylate and methacrylic acid, to which the affinity ligand p-aminophenyl-alpha-D-glucopyranoside was coupled, served as the bifunctional ligand (ligand-Eudragit). Owing to the tetrameric structure of Con A, where each subunit has the ability to bind one sugar moiety, and to the multivalency of ligand-Eudragit, a network was formed between the Con A and ligand-Eudragit. It was possible to detect the initial soluble complexes formed by dynamic laser light scattering (DLLS) long before any precipitate could be analysed by transmittance measurements. The rate of complex formation was highly dependent on the ratio between lectin and ligand-Eudragit. It was further shown that the system did not reach equilibrium within the 110 min studied. When the complex formation was studied in the presence of glucose, the build-up rate was decreased to different degrees depending on the sugar concentration used. At high glucose concentrations the complex formation was completely inhibited.

Affinity Labels↗
PubMed1998

Preliminary studies on the purification of a monoclonal antibody by affinity precipitation with Eudragit S-100.

A simple procedure for the purification of an IgG-type monoclonal antibody by affinity precipitation using Eudragit S-100 is presented. The ligand, a microbial lipase previously used as antigen, was coupled to the polymer at a concentration of 40 mg lipase/g Eudragit. This macroligand was reversibly precipitated by manipulating the pH at values higher and lower than 4.8. The effects of polymer concentration and dilution of hybridoma culture supernatant on the overall precipitation process were evaluated. The best purification factor was achieved with a polymer concentration of 0.1% (w/v) and a supernatant dilution of 1:3. The preliminary studies reported here enabled the purification of a monoclonal antibody in one step with an activity yield (by ELISA) of 50%-55% and a purification factor of ca 6.

Affinity Labels↗
PubMed1998

Protein displacement in dye-ligand chromatography using neutral and charged polymers.

Displacement chromatography was demonstrated to perform separations efficiently under mass-overloaded conditions, offering advantages such as increased product recovery and purity, superior resolving power, and concentration and purification in a single processing step. The use of water-soluble polymers for protein displacement in dye-ligand chromatography was initiated in our laboratory. The polymers for displacement were selected using differences spectroscopy to monitor their interactions with a dye-ligand in solution. Non-charged polymers such as poly(N-vinyl pyrrolidone) and poly(N-vinyl caprolactam) efficiently displaced lactate dehydrogenase from porcine muscle from a Blue Sepahrose column. The latter polymer, being thermosensitive, could be easily removed from the eluate and recovered by precipitation at 45 degrees C and low-speed centrifugation. The positively charged polymer poly(ethylene imine) proved to be an even more efficient displacer. The dye-ligand column could be regenerated after application of displacer either by washing with a solution of the soluble ligand Cibacron Blue (in the case of non-charged polymers) or by washing with highly alkaline solutions containing polyanions (in the case of poly(ethylene imine)) The latter formed a soluble complex with poly(ethylene imine) and stripped the column from the polymer.

Animals↗
PubMed1998

Reactivation of glyceraldehyde-3-phosphate dehydrogenase using conjugates of monoclonal antibodies with polyelectrolyte complexes. An attempt to make an artificial chaperone.

The simplified model of chaperone action when the inactive misfolded forms are removed from the reaction media preventing aggregation was developed using antibodies in combination with polyelectrolyte complexes. The antibodies, which bind specifically inactive dimers of glyceraldehyde-3-phosphate dehydrogenase but not native tetramers, were coupled covalently to poly(methacrylic acid). The treatment of inactivated GAPDH with this conjugate followed by its precipitation after equimolar addition of polycation, poly-(N-ethyl-4-vinylpyridinium bromide), resulted in a significant increase in the specific activity of the enzyme.

Animals↗