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C E Glatz

Publications and source records attributed to C E Glatz.

At least 19 recordsLinked to original sources
PubMed2000

Suitability of immobilized metal affinity chromatography for protein purification from canola.

This work demonstrates that proper selection of a metal ion and chelating ligand enables recovery of a his(6)-tagged protein from canola (Brassica napus) extracts by immobilized metal affinity chromatography (IMAC). When using Co(2+) with iminodiacetate (IDA) as the chelating ligand, beta-glucuronidase-his(6) (GUSH6) can be purified from canola protein extract with almost homogeneous purity in a single chromatographic step. The discrimination with which metal ions bound native canola proteins followed the order Cu(2+) < Ni(2+) < Zn(2+) < Co(2+) in regard to elimination of proteins coeluted with the fusion protein. IDA- and nitrilotriacetate (NTA)-immobilized metal ions showed different binding patterns, whose cause is attributed to a more rigid binding orientation of the his(6) in forming a tridentate with Me(2+)-IDA than in forming a bidentate with Me(2+)-NTA. The more flexible binding allows for multisite interactions over the protein.

Brassica↗
PubMed1998

Contribution of protein charge to partitioning in aqueous two-phase systems.

Protein partitioning in aqueous two-phase systems based on phase-forming polymers is strongly affected by the net charge of the protein, but a thermodynamic description of the charge effects has been hindered by conflicting results. Many of the difficulties could be because of problems in isolating electrochemical effects from other interactions of phase components. We explored charge effects on protein partitioning in poly(ethylene glycol)-dextran two-phase systems by using two series of genetically engineered charge modifications of bacteriophage T4 lysozyme produced in Escherichia coli. The two series, one in the form of charged-fusion tails and the other in the form of charge-change point mutations, provided matching net charges but very different polarity. Partition coefficients of both series were obtained and interfacial potential differences of the phase systems were measured. Multi-angle laser light scattering measurements were also performed to determine second virial coefficients. A semi-empirical model accounting for the roles of both charge and non-charge effects on protein partitioning behavior is proposed, and the results predicted from the model are compared to the results from the experiments.

Bacteriophage T4↗
PubMed1998

Propionic acid production by extractive fermentation. I. Solvent considerations.

Solvent selection for extractive fermentation for propionic acid was conducted with three systems: Alamine 304-1 (trilaurylamine) in 2-octanol, 1-dodecanol, and Witcohol 85 NF (oleyl alcohol). Among them, the solvent containing 2-octanol exhibited the highest partition coefficient in acid extraction, but it was also toxic to propionibacteria. The most solvent-resistant strain among five strains of the microorganism was selected. Solvent toxicity was eliminated via two strategies: entrapment of dissolved toxic solvent in the culture growth medium with vegetable oils such as corn, olive, or soybean oils; or replacement of the toxic 2-octanol with nontoxic Witcohol 85 NF. The complete recovery of acids from the Alamine 304-1/Witcohol 85 NF was also realized with vacuum distillation.

Fermentation↗
PubMed1996

Fed-batch fermentation with and without on-line extraction for propionic and acetic acid production by Propionibacterium acidipropionici.

Fed-batch propionic and acetic acid fermentations were performed in semi-defined laboratory medium and in corn steep liquor with Propionibacterium acidipropionici strain P9. On average, over four experiments, 34.5 milligrams propionic acid and 12.8 milligrams acetic acid were obtained in about 146 h in laboratory medium with 79 milligrams glucose added over five feeding periods. The highest concentration of propionic acid, 45 milligrams, was obtained when the glucose concentration was not allowed to drop to zero. In corn steep liquor 35 milligrams propionic acid and 11 milligrams acetic acid were produced in 108 h from 59.4 milligrams total lactic acid provided as seven feedings of corn steep liquor. Extractive fed-batch fermentations were conducted in semi-defined medium using either flat-sheet-supported liquid membranes or hollow-fiber membrane extraction to remove organic acids from the culture medium. As operated during the course of the fermentation, these systems extracted 25% and 22% of the acetic acid and 36.5% and 44.5% of the propionic acid, respectively, produced in the fermentation. Total amounts of acids produced were about the same as in comparable nonextractive fermentations: 30-37 milligrams propionic acid and 13 milligrams acetic acid were produced in 150 h. Limitations on acid production can be attributed to limited substrate feed, not to failure of the extraction system.

Acetic Acid↗
PubMed1990

Polyelectrolyte precipitation of beta-galactosidase fusions containing poly-aspartic acid tails.

Protein recovery from industrial microbial processes can be very expensive, often exceeding the cost of protein production. We have genetically engineered 3 beta-galactosidase (beta-gal) fusion proteins containing poly-aspartic acid tails to test the effect of the tails on recovery by the relatively inexpensive method of polyelectrolyte precipitation. The fusion proteins, designated T1, T2, and T3, were constructed with C-terminal tails of 5, 11, and 16 aspartic acid residues, respectively. The fusion proteins were expressed in Escherichia coli, and purified by affinity chromatography. T1 and T2 had specific activities similar to that of wildtype beta-gal, whereas the specific activity of T3 was about half that of T1 and T2. The increased net charge of the fusion proteins compared to wildtype beta-gal was indicated both by ion-exchange chromatography and their migration pattern in non-denaturing polyacrylamide gel electrophoresis. All three tails enhanced polyethyleneimine (PEI) precipitation of the fusion proteins compared to wildtype beta-gal. At a low PEI/protein ratio (0.01, g g-1), recovery by precipitation of T2 and T3 was more than 2 X that of the beta-gal control, whereas that of T1 was only slightly greater than that of the control. At a higher PEI/protein ratio (0.03, g g-1) the amount of precipitation of all three fusion proteins was nearly the same, about 1.5 X that of the control.

Aspartic Acid↗
PubMed1990

Separation processes in biotechnology. Precipitation.

Precipitation has a clear role in downstream processing for product concentration, and ongoing developments promise a more important role in fractionation. Its advantages include adaptability to continuous processing and larger scales, the wide variety of possible precipitants, and the ability to retain biological activity. The focus here has been on precipitation of proteins, but many of the principles apply to nucleic acid removal and precipitation of lower-molecular-weight biological products, such as pharmaceuticals.

Biotechnology↗
PubMed1985

The pharmacokinetics of chlortetracycline orally administered to turkeys: influence of citric acid and Pasteurella multocida infection.

A physiologically based pharmacokinetic model was developed to describe the absorption and disposition of chlortetracyline (CTC) in the healthy and diseased (fowl cholera) turkey. The CTC was given (with and without citric acid) as an oral (15 mg/kg) or i.v. (1 mg/kg) dose. When minerals (0.3 g/L Ca2+, 0.1 g/L Mg2+) were dissolved in the bird's drinking water, the model indicated that the addition of citric acid (mass ratio of 10 citrate: 1 CTC) increased the fraction of dose absorbed from 0.06 to 0.16; once absorbed, the fractions of drug eliminated by renal excretion, biliary secretion, and chemical decomposition were 50, 46, and 4%, respectively. The presence of fowl cholera appeared to increase plasma levels by increasing the intestinal permeability and lowering the hepatic and/or renal clearance.

Administration, Oral↗
PubMed1984

Oral absorption of chlortetracycline in turkeys: influence of citric acid and Pasteurella multocida infection.

Plasma and tissue concentrations, following the oral administration of the antibiotic chlortetracycline (CTC) alone or with citric acid, were determined in healthy and infected (Pasteurella multocida) turkeys. The principal results were: 1) The dose (of CTC) versus plasma level relationship was nearly linear. 2) Addition of citric acid to an oral preparation produced significantly higher plasma levels when divalent cations Ca2+ (.3 g/liter) and Mg2+ (.1 g/liter) were present in the drinking water and dosage solution than when citric acid was omitted. 3) The concentration of CTC was considerably higher in the liver and kidney than in the muscle and brain. 4) Birds infected with P. multocida had significantly higher plasma levels than healthy birds. 5) Oral administration of CTC increased the survival rate of the birds infected with P. multocida.

Administration, Oral↗
PubMed1983

Pharmacokinetics of chlortetracycline potentiation with citric acid in the chicken.

Serum concentrations of chlortetracycline (CTC) in healthy chickens were determined for the 24-hour period after they were given CTC (with and without citric acid) as an oral (25 mg/kg) or IV (0.9 mg/kg) dose. The oral time-course drug data were fitted adequately by a 2-compartment pharmacokinetic model with absorption. The resulting absorption rate constant (Ka) for the birds orally given CTC with citric acid was nearly equal to that for the birds given CTC alone. Although the uptake of orally administered CTC was rapid, only a small fraction of the dose was absorbed. The administration of citric acid-CTC significantly increased the mean serum concentration of CTC and the fraction of the dose absorbed. The citric acid-CTC mixture also produced significantly higher elimination (Kel) and distribution (K12) rate constants for CTC.

Administration, Oral↗
PubMed1979

The kinetics of binding of serum lipoproteins by immobilized heparin.

A model arterial system of heparin immobilized on an agarose gel was used to study the amount and kinetics of binding of porcine serum lipoproteins to heparin. Binding occurred to lipoproteins in the density range 1.006 less than d less than 1.062, but there was no binding with high density lipoprotein. A theoretical model of the kinetic experiments was formulated and used to demonstrate that the rate of the binding reaction could be considered instantaneous relative to the rate of transport of lipoproteins. Extrapolation of these results to arterial levels of glycosaminoglycans and lipoprotein indicate that complexes of lipoprotein and the glycosaminoglycans could account for much of the cholesterol entrapment in atherosclerotic lesions.

Animals↗
PubMed1979

Distribution of glycosaminoglycans in consecutive layers of the rabbit aorta.

Transmural variations in various glycosaminoglycan (GAG) fractions were determined in adventitia-free thoracic aortas from rabbits. Total glycosaminoglycan concentration decreased from intima to outer media. These data are similar to total GAG concentration in bovine and human aortas as reported by others. There is a marked decrease in the concentration of the combined chondroitin sulfate-dermatan sulfate component with increasing distance from the endothelial surface. These transmural differences are linked to the possible variation of the diffusion coefficient of a diffusing solute as a function of distance, which can affect the concentration profile of the solute.

Animals↗
PubMedDate not supplied

Influence of glycosaminoglycan content of mass transfer behavior of porcine artery wall. Part 1. Diffusive transport of 45Ca2+ and 3HHO.

The diffusion coefficients of Ca2+ and H2O in in vitro porcine arterial tissue were determined to provide a measure of vascular permeability. The effect of anatomical location and diet on the magnitude of that permeability was evaluated. The diffusion coefficient of calcium was found to vary focally and, in addition, pulmonary artery was much more permeable than thoracic aorta. Four months feeding of a high lipid diet did not affect diffusive transport.

Animals↗
PubMedDate not supplied

Influence of glycosaminoglycan content on mass transfer behavior of porcine artery wall. Part 2. Differences in mass transfer rates related to variations in glycosaminoglycan content.

It has been suggested that the glycosaminoglycans (GAG) influence atherogenesis by regulating the permeability of the arterial wall. For this reason, a study has been made of the diffusive transport of Ca2+ and water across the in vitro porcine artery wall, where particular attention was focused on the influence of GAG content and distribution within the wall on the transport properties. The radioisotop-s 45Ca and 3HHO were used to measure the tracer-diffusion flux in a stirred, two chamber diffusion cell. GAG were isolated, fractionated using a cetyl pyridinium chloride-cellulose column procedure, and assayed using a colorimetric carbazole reaction for uronic acid. The biochemical analyses showed that the pulmonary artery contains significantly more hyaluronic acid and dermatan sulfate than found in two locations in the thoracic aorta. In addition, a significant regression was found for the diffusion coefficient of 45Ca2+ (99% level) and 3HHO (95% level) versus specific GAG fractions. The regression indicated an increase in permeability with increase in the ratio of sulfated: nonsulfated GAG.

Animals↗
PubMedDate not supplied

Fusion tails for the recovery and purification of recombinant proteins.

Several fusion tail systems have been developed to promote efficient recovery and purification of recombinant proteins from crude cell extracts or culture media. In these systems, a target protein is genetically engineered to contain a C- or N-terminal polypeptide tail, which provides the biochemical basis for specificity in recovery and purification. Tails with a variety of characteristics have been used: (1) entire enzymes with affinity for immobilized substrates or inhibitors; (2) peptide-binding proteins with affinity to immunoglobulin G or albumin; (3) carbohydrate-binding proteins or domains; (4) a biotin-binding domain for in vivo biotination promoting affinity of the fusion protein to avidin or streptavidin; (5) antigenic epitopes with affinity to immobilized monoclonal antibodies; (6) charged amino acids for use in charge-based recovery methods; (7) poly(His) residues for recovery by immobilized metal affinity chromatography; and (8) other poly(amino acid)s, with binding specificities based on properties of the amino acid side chain. Fusion tails are useful at the lab scale and have potential for enhancing recovery using economical recovery methods that are easily scaled up for industrial downstream processing. Fusion tails can be used to promote secretion of target proteins and can also provide useful assay tags based on enzymatic activity or antibody binding. Many fusion tails do not interfere with the biological activity of the target protein and in some cases have been shown to stabilize it. Nevertheless, for the purification of authentic proteins a site for specific cleavage is often included, allowing removal of the tail after recovery.

Amino Acids↗
PubMedDate not supplied

Precipitation of nucleic acids with poly(ethyleneimine).

Removal of nucleic acids from cell extracts is a common early step in downstream processing for protein recovery. We report on the precipitation of nucleic acids from a homogenate of Saccharomyces cerevisiae by addition of the cationic polyelectrolyte poly(ethyleneimine) (PEI), focusing on the effect of PEI dosage on particle size, protein loss, and extent of nucleic acid removal in both batch and continuous mode. Better than 95% removal of nucleic acids from yeast homogenates was achieved by means of precipitation with PEI with protein losses of approximately 15% with or without previous removal of cell debris. The coprecipitated protein is predominately large molecular weight material and exhibits both low and high isoelectric points. Such treatment does not aggregate the cell debris; size distribution of the precipitated particles from a continuous precipitator is very similar to that for protein precipitation.

Chemical Precipitation↗
PubMedDate not supplied

Genetic engineering strategies for purification of recombinant proteins from canola by anion exchange chromatography: an example of beta-glucuronidase.

The elution behavior of native canola proteins from different anion-exchange resins was determined. The elution profiles showed the potential for simplified recovery of acidic recombinant proteins from canola. When Q-sepharose fast flow was used, there were three optimal salt elution points at which a recombinant protein would have minimal contamination with native proteins. The feasibility of exploiting this advantage was examined for recovery of the acidic protein beta-glucuronidase (GUS/GUSD0 from the Escherichia coli gene) along with three polyaspartate fusions to the wild-type GUS. The fusions contained 5 (GUSD5), 10 (GUSD10), or 15 (GUSD15) aspartic acids fused to the C-terminus and were chosen to extend the elution time. The three fusions and the wild-type enzyme were produced in E. coli, purified, and added to canola extracts before chromatography. The equivalence of this spiking experiment to that of extracting a recombinant protein from transgenic canola was determined in a control experiment using transgenic canola expressing the wild-type enzyme. Behavior in the transgenic and spiked experiments was equivalent. GUSD0 eluted at the earliest optimal elution point; the addition of polyaspartate tails resulted in longer retention times and better selective recovery. If one assumes binding through a single fusion (the protein is a tetramer), there is a nearly linear shift in elution within the salt gradient of 17 mM per added charge up to 10, with a reduced increment from 10 to 15. The fusions and their enzymatic activity proved very stable in the canola extracts through 7 days in cold storage, providing flexibility in process scheduling.

Anion Exchange Resins↗
PubMedDate not supplied

Characterization and polyelectrolyte precipitation of beta-galactosidase containing genetic fusions of charged polypeptides.

Genetically engineered versions of beta-galactosidase were constructed through the addition of charged polypeptide fusion tails for the purpose of enhancing polyelectrolyte precipitation. Negatively charged aspartic acid tails and positively charged poly(arginine) tails were added to beta-galactosidase from Escherichia coli. These fusion proteins were all shown to possess specific activity equal to that of the native enzyme. Gel permeation and ion-exchange chromatography provided evidence concerning the integrity of the tails as well as their altered charge characteristics. All enzymes containing charged tails displayed enhanced polyelectrolyte precipitation over the native enzyme. An optimal number of charged residues, beyond which no further enhancement of precipitation was observed, was found to be approximately 10 residues for each type of tail. No interference from nucleic acids was observed in the precipitation of positively tailed beta-galactosidase.

Acrylic Resins↗