Fractional Protein Precipitation Research Articles

Fractional Precipitation of Proteins by Ammonium Sulfate

Fractional Precipitation of Proteins by Ammonium Sulfate

Effect of Agitation Intensity on Protein Solubility Behavior

AbstractA biochemical engineering framework to study fractional protein precipitation has been developed. For optimization, design and safe operation of the fractional protein precipitation process, the effects of process variables must be known. The solubility behavior of trypsin from bovine pancreas, as a function of ammonium sulphate concentration has been examined. The effect of agitation speed on enzyme and total protein solubility and enzyme specific activity has been investigated.

A Kalman filter algorithm and monitoring apparatus for at-line control of fractional protein precipitation

Downstream processing operations are often carried out blind in the process timescale since product monitoring on-line is not common. Knowledge of the location and concentration of the product and key contaminants is complementary to other process information for process development and, if available on-line in conjunction with a suitable model, control. This article sets out to demonstrate a model describing a two-cut fractional protein precipitation process and how this may be used for control of the process to maximize yield in the face of variable process stream conditions. Estimation of the model parameters is achieved by means of data-fitting by least squares and in comparison prediction by a Kalman filter algorithm. A description and error analysis of equipment for at-line monitoring of the soluble product in a pilot plant environment is presented which includes a micro-centrifuge necessary to clarify small volumes of sample prior to analysis. Finally, an account of the successful implementation of this equipment and the Kalman filter algorithm for control at bench scale is given where conditions in the process stream are deliberately disturbed to test the control operation. (c) 1997 John Wiley & Sons, Inc.

Computer-based simulation of the recovery of intracellular enzymes and its pilot-scale verification

Accurate and reliable models are required for a range of unit operations if simulations are to be used for accelerating the design and optimisation of bioprocesses. This paper presents results of pilot-plant studies that have been used to verify process simulations for a sequence of operations comprising of cell disruption, fractional protein precipitation and centrifugal separation. These have been tested using the purification of the enzyme alcohol dehydrogenase (ADH) from Saccharomyces cerevisiae as being representative of the recovery of a labile intracellular enzyme. Comparison of pilot-plant against simulated data highlights where improvements to the models are required and has resulted in increased confidence in the simulations for a wide range of conditions including the operational scale and the nature of the starting material. Results demonstrate the effectiveness of the verification approach for the development of reliable predictive models to assess the feasibility of process designs and performance of a train of bioprocess operations.

Avaliacao das atividades hemaglutinantes e de interacao com linfocitos T humanos nas fracoes parcialmente purificadas da lectina de massas de ovas de Biomphalaria glabrata (variedade albina)

The extraction and fractional precipitation of proteins from egg masses of the snail Biglabrata (var. albin), with ammonium sulfate, produced a 30-70% saturation fraction that was able to interact with human T lyrnphocytes, besides the original hemagglutinating lectinic activity.The fraction was submitted to chromatography on DEAE-Sepbadex A 50-120 and three further fractions were obtained with distinct biological activities: F-I) hemagglutinating fraction; F-III) Iymphocyte interaction fraction and F- II) fraction with both activities. Lyrnphocyte interaction (LlF) has revealed to be a potent inhibitor of spontaneous E rosette formation with T lymphocytes. Such inhibition was not affected by Ca' and Mg ions and was no reversed in the prescnce of preferencial inhibitors of hemagglutination (ceto-hexoses) or other sugars. Further more, rosette fonnation was not inhibited when sheep red blood cells were previously incubated with LlF.Indirect Immunofluorescence assays and flow cytornetry have demonstrated that LIF blocked the binding of the anti-CD2 monoclonal antibody to lhe lymphocyte membrane receptor, strengthening preliminary data obtained with E rosette inhibition assays.

Pilot-scale verification of a computer-based simulation for fractional protein precipitation

The development and experimental verification at pilot scale of a suite of models for the batch precipitation by two-cut ammonium sulphate salting-out of total protein and alcohol dehydrogenase from yeast homogenate is presented. The model consists of two elements: protein and enzyme solubility prediction and precipitate phase particle property prediction. An isotherm-based approach has been used successfully to describe solubility behaviour for a range of operating conditions typical of those obtained at a process scale. Estimation of the precipitate phase particle size distributions has been achieved through a discretized population balance approach using simplified terms to account for particle breakage and aggregation. The developed model accounts for the effects of average shear rate and residence time in the precipitation vessel across a two orders of magnitude range of scale. The size-density relationship for the precipitate phase has been defined. Results of simulations are compared with pilot scale verification data to confirm the validity of the models developed.

Pilot-scale verification of a computer-based simulation for fractional protein precipitation

Pilot-scale verification of a computer-based simulation for fractional protein precipitation

Copper affinity precipitation as an initial step in protein purification

The affinity of copper ions for histidine residues can be utilized to carry out fractional precipitation of proteins. Using this approach it is possible to purify concanavalin A by a simple two step procedure. The fold purification obtained was 4.5 fold.

A new biochemical engineering approach to the fractional precipitation of proteins

A biochemical engineering framework for optimizing the design and operation of fractional protein precipitation has been developed. The method utilizes a fractionation diagram to represent the purification of a product protein relative to total contaminating protein. The purification factor for a single or double-cut fractional precipitation is obtained as the gradient of an appropriate operating tie-line. A computer algorithm has been devised to maximize the tie-line gradient for a given yield enabling a plot of optimum purification factor versus yield to be constructed. The recovery of the enzyme alcohol dehydrogenase from clarified bakers homogenate using saturated ammonium sulphate has been examined. Fractionation and purification versus yield diagrams were used to investigate the effects of such process parameters as pH, temperature, and initial total protein concentration on fractionation efficiency. The results are discussed in terms of the underlying solubility and mixing phenomena and the industrial application of fractional precipitation.

Purifying hepatitis B surface antigen from recombinant cell cultures by separating cells, fractional protein precipitation of the filtrate and purification on an anion exchanger

Purifying hepatitis B surface antigen from recombinant cell cultures by separating cells, fractional protein precipitation of the filtrate and purification on an anion exchanger

Concentration of Poliomyelitis Virus by Ultrafiltration.

The obvious desirability of finding a means for the more adequate concentration of any of the filterable viruses, led us to attempt to adapt the ultrafiltration method, used by Seibert in the study of tuberculin, to the concentration of poliomyelitis virus. That several of the well studied filterable viruses can be retained by filters or ultrafilters has been known for some time. But the work has been done largely for the purpose of determining the size of the viruses. Krueger and Schultz and Clifton, Schultz and Gebhardt, for example, have made careful studies of the filterability of poliomyelitis virus through graded collodion membranes. Their results indicate that the size of this virus is certainly less than 50 millimicrons in diameter and probably below 25 millimicrons. We have previously employed distillation in vacuo and fractional protein precipitation (Clark, Schindler and Roberts and Clark, Roberts and Preston) for the successful purification and concentration of poliomyelitis virus. Both of these methods, however, necessitate the subsequent elimination of the large amounts of salts by dialysis, before the material can be used for animal inoculation. The method here presented avoids that difficulty and we hope that it may give a more adequate basis for the study of the properties of poliomyelitis and other viruses. Dried soluble cotton was dissolved in concentrations of 8, 10, and 12% in glacial acetic acid and allowed to age, as a rule, at least a month, before being used to form the membranes over the alundum shells of medium porosity. Sterile distilled water was employed for washing the membranes, subsequently testing for the presence of acid with Congo Red. At least a liter of water was run through the membrane after the test for acid became negative. The permeability of the membranes was determined by their capacity to hold back Congo Red, a solution of crystallized tgg albumin and a solution of hemoglobin. The Congo Red was tested for by the addition of acid to the ultrafiltrate, and the presence or absence of tgg albumin was determined by using a specific precipitating serum of high titre. The increasing density of the red color in the “concentrate,” when a small amount of laked blood had been added to the material to be filtered, and the colorless character of the ultrafiltrate served as an indicator of value. Conclusions. A method for the ultrafiltration and concentration of poliomyelitis virus is described. This should be applicable to the further study of the properties of poliomyelitis and other filterable viruses. The method does not result in a direct proportional volumetric increase in the potency of the ultraresidue. A concentration of an inhibiting agent is suggested.