New methods of protein purification. Affinity ultrafiltration.

This review describes a recently developed method for protein purification-affinity ultrafiltration. In affinity ultrafiltration, the protein to be purified is complexed with a macroligand composed of a soluble polymer or an insoluble microparticle with covalently bound, target protein-specific affinity ligands. The complex is trapped by an ultrafiltration membrane, whereas unwanted proteins pass through the membrane. The unwanted proteins are removed from the system by the carrier liquid. The system is then supplemented with an agent eluting the target protein by dissociating it from the microligand complex. The purified protein then passes the membrane, while the macroligand is trapped by it. The macroligand can be re-used after regeneration. Affinity ultrafiltration has a number of advantages over other protein purification techniques: 1) commercial availability of ultrafiltration systems with various high-productivity designs; 2) availability of presynthesized macroligands, which can be supplemented with additional, easily manufactured, commercial latex-based macroligands; 3) rapid separation of large solution volumes; 4) repeated use of equipment, enabling consecutive purification of different proteins; 5) simple scale-up and automation procedures.     

Mechanism of bovine serum albumin aggregation during ultrafiltration

Protein fouling is a critical problem for ultrafiltration. In this study, we adopted bovine serum albumin (BSA) as a model protein and polysulfone membrane as a typical ultrafiltration membrane. We then investigated the factors of the protein denaturation and aggregation, such as stirring shear stress and intermolecular exchange of disulfide during ultrafiltration, and discussed the BSA fouling mechanism. Fourier transform-infrared analysis revealed that magnetic stirring did not cause any difference in the secondary structural change of BSA gel-like deposits on the ultrafiltration membrane. BSA aggregates were collected from BSA gel-like deposits on the ultrafiltration membrane by centrifugation. Polyacrylamide gel electrophoresis in SDS analysis of BSA aggregates proved that the major binding of the BSA aggregates involved intermolecular disulfhydryl binding and that capping the free thiol group in BSA molecules with cysteine induced a remarkable decrease in the amount of the BSA aggregates during ultrafiltration. We concluded that one of the main factors in the BSA aggregation during ultrafiltration is the intermolecular exchange of disulfide through cysteinyl residue. We also found that the BSA aggregation caused a decrease in alpha-helix from 66% to 50% and an increase in beta-sheet from 20% to 36%, which was presumably because the cysteine residues associated with the intermolecular disulfide bonds had been located in alpha-helices. Copyright John Wiley & Sons, Inc.     

Estimation of protein-ligand binding parameters from contiuous ultrafiltration results.

A method of analyzing the result of continuous ultrafiltration experiments to obtain protein-ligand binding parameters is presented. This method employs a nonlinear least-squares regression algorithm coupled with a model of protein-ligand binding which alloww the computation of free ligand concentrations, and a second-order Runge-Kutta method to integrate free concentrations with respect to collected ultrafiltrate. The approach is general and effectively removes the constraints on maximum fraction size imposed by other methods.     

An ultrafiltration assay for nucleotide binding to ribonucleotide reductase

Direct partition through ultrafiltration was applied to develop a method for the study of nucleotide binding to ribonucleotide reductase from Escherichia coli. The assay involved a 0.5- to 1-min centrifugation step where bound and unbound nucleotides are separated over an ultrafiltration membrane. No effects were seen due to hyperconcentration of protein at the membrane surface. The method was verified by measuring binding of dATP, ATP, dTTP, dGTP, and GDP at 25 and 4 degrees C with dissociation constants ranging from 0.1 to 80 microM. The results were in good agreement with earlier data obtained by other techniques and extend our knowledge in the case of ATP and dGTP binding at 25 degrees C.     

Retention of small charged impurities during ultrafiltration

Ultrafiltration is used to remove small impurities from a variety of processing streams. However, the clearance of small charged impurities may be inadequate due to electrostatic exclusion by the charged ultrafiltration membranes, an effect that has been largely unappreciated. Ultrafiltration experiments were performed to evaluate the transmission of several model impurities with different electrical charge through ultrafiltration membranes having different surface charge characteristics. Highly charged impurities are strongly rejected by charged cellulose and polyethersulfone membranes even though these solutes are much smaller than the membrane pore size. These effects could be eliminated by using high ionic strength solutions to shield the electrostatic interactions. The sieving data are in good agreement with model calculations based on the partitioning of charged spheres into charged cylindrical pores. Guidelines are developed for estimating conditions needed to obtain effective removal of small charged impurities through charged ultrafiltration membranes.     

Recovery of biological materials through ultrafiltration

Experiments were performed on a cellulose acetate ultrafiltration membrane (HF-200, ABCOR Inc., Cambridge, Mass.) to test its efficacy in concentrating and purifying a crude enzyme (trypsin) preparation. Studies were also made to determine the influence of inorganic salts, pressure, and temperature on the rate of ultrafiltration for this membrane. The results showed reductions in the rates will be encountered due to the presence of inorganic salts. However, the reduced rates were still sufficiently high to make this method extremely attractive. Operating at filtration pressures above 75 psi at, 20 to 30°C for this membrane does not show any beneficial effect in terms of ultrafiltration rates. However, at 10°C there were continual increases in the filtration rates up to 100 psi. Concentration and purification studies with trypsin yielded a concentration factor of 8.35 and a purification factor 2.35. It was shown concretely that the purification of the enzyme was due to the passage of low molecular weight proteins (below 20,000) through the membrane. Enzyme activity slightly greater than 90% was obtained: 70% was found in the concentrate and 20% in the filtrate. It is concluded that membrane ultrafiltration is an ideal simple, rapid, and economical method for the recovery of biological active substances.     

FACTORS REQUIRED FOR Ca-SENSITIVE ACETYLCHOLINE RELEASE FROM CRUDE SYNAPTIC VESICLES

Abstract— Studies were made on intracellular factors required for the release of ACh from crude synaptic vesicles prepared from rat brain. A factor stimulating ACh release in the presence of calcium ions was found in the cell sap.
Cell sap obtained by hypotonic shock of a crude mitochondrial fraction stimulated ACh release from the synaptic vesicles in the presence of ATP and magnesium ions. Calcium ions were essential for its effect and the optimum concentration of calcium was about 10⁻⁴ m . The stimulatory factor in cell sap passed through a cellulose or collodion membrane or ultrafiltration membrane UM 10, but not through that of an ultrafiltration membrane UM 05. The factor was not affected by trypsin or α-chymotrypsin, but was inactivated by pronase or carboxypeptidase-A. Thus it seemed to be a peptide-like substance with a rather low molecular weight. Cyclic-AMP, cyclic-GMP, colchicine and vinblastne did not affect ACh release under our experimental conditions.     

STATISTICAL EVALUATION OF SIEVE CONSTANTS IN ULTRAFILTRATION

The partial retention of the disperse phase in the ultrafiltration of a monodisperse system through an isoporous filter is interpreted on a statistical basis, and a simple expression for the sieve constant is evaluated in terms of the calibrated membrane porosity and the particle size. Curves calculated from this expression are in reasonable agreement with experimental data for the ultrafiltration of serum albumin, hemocyanin (Helix), and foot-and-mouth disease virus.     

Application of ultrafiltration for enzyme retention during continuous enzymatic reaction

In most enzymatic reactions, batch or continuous, separation of the enzyme for reuse is difficult if not impossible. A process will be presented in which an Ultrafiltration membrane serves to separate the reaction products from the enzyme and the substrate. In this manner the enzyme may be retained and re-used. Furthermore, under these conditions, the enzyme need only be present in catalytic amounts regardless of the amount of product produced. Under proper operating conditions and proper ultrafiltration membrane selection, a pure solution of α-amylase from Bacillus subtilis may be retained with no loss in enzyme activity over a test period of 30 hr after steadystate has been achieved. In the presence of substrate, the membrane support and ultrafiltration cell serve as the reaction vessel for the hydrolysis of starch. The substrate is continuously pumped into the cell under constant ultrafiltration pressure. The di-, oligo-, and polysaccharides formed from the enzyme reaction then either pass through the membrane as products or are retained. The molecular weight distribution of the products is dependent on the nominal molecular weight cut-off of the membrane, absolute ultrafiltration pressure, enzyme-to-substrate ratio, temperature, and residence time of the substrate in the reactor. In addition to the partial hydrolysis of starch by α-amylase, some preliminary findings on the complete hydrolysis of starch by glucoamylase will also be presented. In these latter studies, the substrate may be completely hydrolyzed to glucose units.     

Cleaning of inorganic membranes after whey and milk ultrafiltration

Cleaning of an inorganic ultrafiltration membrane has been quantified through hydraulic, physicochemical, and spectroscopic (infrared and x-photoelectron spectroscopy) analyses. An efficient cleaning sequence of nitric acid followed by sodium hypochlorite has been proposed for cleaning of defatted whey protein concentrate and milk ultrafiltration membranes. The influence of reversed sequence and time reduction are discussed together with the action of both cleaning chemicals. In spite of residual fouling left after every cleaning sequence studied, hydraulic cleanliness of the membrane was achieved, particularly after the standard procedure.     

New system for preparative electrochromatography of proteins

Electrochromatography employs an axial electric field across a chromatographic stationary phase to separate proteins and other molecules based on differences in electrophoretic mobility. Because the separation is electrically driven, the need for additional chemical reagents is reduced. Two major impediments to scale-up of electrochromatography columns, removal of heat and electrolysis gases, have historically limited the diameter of packed columns to 2.5 cm ID with volumes of approximately 55 mL. We report a novel electrochromatography column that effectively removes electrolysis gases and minimizes heating. A vital component of this system is a new electrode design that couples a platinum gauze with an ultrafiltration membrane across both ends of the column. Use of a methacrylate base stationary phase enabled axial voltage gradients of 10 to 20 V/cm. Thermocouples inserted radially in the column at four axial positions showed that the flow of a 4 degrees C mobile phase coupled with heat conduction through the column walls controlled the temperature to 28 degrees C. The new column design, with dimensions of 3.81 cm ID x 38.1 cm long and bed volume of 400 mL, was demonstrated by separating mixtures of BSA and myoglobin. The column was operated in a horizontal position with radial sample injection and withdrawal at the ends of the packed bed. These experiments are a first step in demonstrating that scale-up of electrochromatography columns can be achieved by choosing appropriate flow rates, voltage gradients, and stationary phase.     

Effect of ion binding on protein transport through ultrafiltration membranes

Electrostatic interactions can have a significant impact on protein transmission through semipermeable membranes. Experimental data for the transport of bovine serum albumin (BSA) through a polyethersulfone ultrafiltration membrane were obtained in different salt solutions over a range of pH and salt concentrations. Net BSA charge under the same conditions was evaluated from mobility data measured by capillary electrophoresis. The results show that specific ionic composition, in addition to solution pH and ionic strength, can strongly affect the rate of protein transport through semipermeable ultrafiltration membranes. The effects of different ions on BSA sieving are due primarily to differences in ion binding to the protein, which leads to significant differences in the net protein charge at a given pH and ionic strength. This effect could be described in terms of an effective protein radius, which accounts for the electrostatic exclusion of the charged protein from the membrane pores. These results provide important insights into the nature of the electrostatic interactions in membrane systems.     

Role of some whey components on mass transfer in ultrafiltration

Ultrafiltration through Carbosep M(4) mineral membrane of protein solutions of decreasing complexity (whey before and after centrifugation or clarification, beta-lactoglobulin) was studied. Mathematical models were used to explain variations in flux with time. Taking into account variations in protein retention and hydraulic resistance of the membrane during ultrafiltration, proteins and lipoproteins were found to be involved not only in the polarization layer (reversible fouling leading to a difference in the osmotic pressure), but also in irreversible fouling by adsorption. Morever, the presence of particles (e.g., inorganic precipitates) in whey explains the build-up of a deposit over and within the membrane which contributes to the decline in flux after 1 h ultrafiltration. The relative importance of these phenomena was quantified.     

A new conception of enzymatic membrane reactor for the production of whey hydrolysates with low contents of phenylalanine

A novel concept of enzymatic membrane reactor (EMR) is presented, aiming at the production of cheese whey protein hydrolysates with low contents of phenylalanine (Phe) for the diet of phenylketonuria patients. Whey proteins were first hydrolyzed by chimotrypsin, followed by the action of carboxypeptidase A (CPA), immobilized on agarose gel particles, which were retained inside the reactor using a filter. The liquid medium passes through hollow fiber ultrafiltration unit (1 kDa cut-off), and the retentate is recycled to the reactor. The innovation here is that the membrane is not used to retain the enzyme neither physically nor as an immobilization support. The EMR provided a higher performance than the classical, sequential approach: batch reaction followed by ultrafiltration. It was confirmed that the removal of products promoted by the EMR enhances reaction rates, due to the reduction of inhibition effects. A mathematical model of the EMR is also presented and validated.     

Spinach Chloroplast Membranes after Differential Lipid Extraction

Chloroplasts from spinach were fixed in glutaraldehyde and extracted with three different lipid solvents, after which the lipid composition was analyzed. Studies were also made with the electron microscope. In cold dry acetone, which removes 75 % of lipids, the basic structure of the membranes is unchanged. Acetone with 10 % water removes 89 % of the lipids and a mixture of chloroform with methanol removes 93 % of the lipids, both solvents leaving nearly unrecognizable membrane structures. The relationship between lipid composition and membrane structure is discussed.     

The purification of recombinant hepatitis B surface antigen by hollow fibre ultrafiltration membrane diafiltration

Summary Recombinant hepatitis B surface antigen (HBsAg) broth was purified by diafiltration with a hollow fibre ultrafiltration membrane (cut off 100 kDa), by pre—treating the HBsAg broth with enzyme, most of the contaminating proteins was removed and very high recovery ratio of HBsAg was achieved. When HBsAg solution was concentrated using hollow fibre ultrafiltration membrane to 10% of its original volume, the HBsAg was recovered almost completely. Accordingly, membrane purification of HBsAg is a high yield, fast method.     

Deproteination of nucleic acids by filtration through a hydrophobic membrane.

To remove or inactivate an enzyme from DNA in multistep procedures in molecular biology, it is often necessary to phenol extract the solution, followed by chloroform extraction and ethanol precipitation. In addition to being time-consuming and hazardous, there can be significant loss of DNA with this procedure, especially when small volumes or amounts of DNA are being used. We have found that filtering analytical reaction mixtures through a hydrophobic membrane specifically to remove protein is a rapid alternative to phenol extraction. Within broad limits commonly encountered in molecular biology, filtration through a polyvinylidene difluoride membrane quantitatively removes a variety of enzymes without significant loss of double-stranded nucleic acid.     

Surfactant pretreatment of a polysulfone ultrafilter for reduction of antifoam fouling

Effectiveness of surfactant precoat treatment of the polysulfone ultrafilter was first investigated for reduction of membrane fouling in ultrafiltration of antifoam. Fifteen different surfactants, including alcohols and synthetic nonionic surfactants, were tested. In general, pretreatment with nonionic surfactant gave a larger flux than that with alcohol did. The flux increase by pretreatment with nonionic surfactant depended on a hydrophile lipophile balance (HLB) value and type of hydrophobic tail. The most effective surfactant for reducing antifoam fouling among the 15 surfactants was Brij-58 which has an HLB value of 16 and a straight alkyl hydrophobic chain. The ultrafiltration flux of the membrane treated with Brij-58 was almost three times larger than that of untreated membrane. The precoat treatment with Brij-58 was the most effective for reducing antifoam fouling in terms of rejection properties.Furthermore, flux was also improved by the surfactant pretreatment in ultrafiltration of model process streams, such as fermentation media, broth, and yeast suspension with or without antifoam. The surfactant Brij-58 was found to be more effective for reducing membrane fouling in ultrafiltration of model stream YG compared with ethanol or Brij-35. The mean flux increase by the pretreatment with Brij-58 was about 80% in ultrafiltration of the model stream without antifoam. When antifoam was added to the model stream, flux was almost doubled by the pretreatment with Brij-58. The effectiveness of surfactant precoat treatment for reducing membrane fouling was also confirmed in terms of rejection properties. (c) 1994 John Wiley & Sons, Inc.     

Transport and kinetics in sandwiched membrane bioreactors.

A bioreactor in which living yeast cells are sandwiched between an ultrafiltration membrane and a reverse osmosis membrane was constructed, and experiments were performed for the conversion of substrate glucose to product ethanol. A set of equations that include both transport through a series of barrier layers and bioreaction rate were developed to predict the performance of the sandwich bioreactor. The above equations were solved by using numerical values for the transport parameter and the bioreaction rate constant, and the results are compared with the experimental data.     

Enzymatic hydrolysis of sodium caseinate in a continuous ultrafiltration reactor using an inorganic membrane

Continuous hydrolysis of sodium caseinate by alcalase was investigated in a recycle bioreactor coupled to an inorganic M5 membrane module. The effects of various substrate concentrations and the role of an ultrafiltration membrane on conversion rate were reported. Although a high level of conversion was obtained in the retentate side at a steady state, only part of the products formed was transmitted through the inorganic membrane. Degree of hydrolysis and product concentration in the reactor seem to be the main factors limiting product output during the continuous hydrolysis.