Oxidative renaturation of hen egg-white lysozyme in polyethylene glycol-salt aqueous two-phase systems.

Aqueous two-phase systems have been widely used for the separation and concentration of proteins. In this work we investigated the possibility of using aqueous two-phase system for the renaturation of inclusion body proteins by studying the effect of polyethylene glycol (PEG)-salt systems on the oxidative renaturation of hen egg-white lysozyme (HEWL) with guanidinium chloride (GdmCl) present in the system. To accomplish phase separation at moderately low concentrations of polymer and salt, the total GdmCl concentration had to be kept low (<1 M). The unfolded protein exhibited very low solubility under these conditions. In an attempt to increase the solubility of the protein, temperatures of 40, 50, and 60 degrees C were investigated. The effect of PEG molecular weight was also addressed. Best renaturation yields were obtained when using PEG 3400 and working at 50 degrees C. However, the total protein concentration had to be kept at a low level of 0.2 mg/mL. Lowering the total GdmCl concentration in the system resulted in increased aggregation.     

Effect of poly(ethylene glycol) on the Ca2+-induced fusion of didodecyl phosphate vesicles

This paper reports a study of the effect of the dehydrating agent poly(ethylene glycol) (PEG) on didodecyl phosphate (DDP) bilayers and on the fusion activity of DDP vesicles as a function of the molecular weight of PEG. PEG 8K in a concentration of 10 wt % does not induce fusion. However, Ca2+-induced fusion is promoted as reflected by a lowering of the Ca2+ threshold concentration. This effect can most likely be attributed to the dehydrating capacity of the polymer. Interestingly, low concentrations (0.1 wt %) of PEG 20 K induce a moderate fusion capacity. At higher concentrations (0.5 wt %) fusion is inhibited, irrespective of the presence of Ca2+. These molecular weight dependent effects can be rationalized by taking into account that the clouding temperature differs for PEGs of different molecular weights. In the case of PEG 20K a microscopic phase separation will occur at the bilayer-water interface because PEG-PEG interactions and presumably PEG-DDP interactions are favored over PEG-water interactions. As a consequence, the DDP vesicle surface becomes covered with PEG 20K, resulting in a steric stabilization of the vesicles. This will impede or prevent, depending on the polymer concentration, the vesicles from approaching each other sufficiently close for fusion to occur.     

Ultrafiltration characteristics of pegylated proteins

There is growing clinical interest in the use of pegylated recombinant proteins with enhanced stability, half-life, and bioavailability. The objective of this study was to develop a quantitative understanding of the ultrafiltration characteristics of a series of pegylated proteins with different degrees of pegylation. Sieving data were compared with available theoretical models and with corresponding results for the partition coefficient in size exclusion chromatography (SEC). The sieving coefficients of the pegylated proteins depended not only on the protein size and the total molecular weight of the polyethylene glycol (PEG) but also on the number of PEG chains. This is in sharp contrast to the partition coefficient in SEC, which was uniquely determined by the total molecular weight of the PEG and protein. This difference is due to the deformation and/or elongation of the PEG chains caused by the convective flow into the membrane pores, an effect that is not present in SEC. These results provide important insights into the transport and separation characteristics of pegylated proteins.     

Phase separation of Triton X-100 micelle solution induced by osmotic stress

We have found out that the phase separation of Triton X-100 micelle solution was caused by the addition of poly(ethylene glycol) (PEG) above a critical concentration. The critical concentration of PEG depended on its molecular weight and temperature, larger molecular weight or higher temperature giving lower critical concentration. These results were analyzed on the basis of the osmoelastic coupling theory recently proposed by us (Biochemistry (1989) 28, 3710-3715; Biochemistry (1989) 28, 5626-5630).     

Correlation for the partition behavior of proteins in aqueous two-phase systems: effect of surface hydrophobicity and charge

Correlations to describe the effect of surface hydrophobicity and charge of proteins with their partition coefficient in aqueous two-phase systems were investigated. Polyethylene glycol (PEG) 4000/phosphate, sulfate, citrate, and dextran systems in the presence of low (0.6% w/w) and high (8.8% w/w) levels of NaCl were selected for a systematic study of 12 proteins. The surface hydrophobicity of the proteins was measured by ammonium sulfate precipitation as the inverse of their solubility. The hydrophobicity values measured correlated well with the partition coefficients, K, obtained in the PEG/salt systems at high concentration of NaCl (r = 0.92-0.93). In PEG/citrate systems the partition coefficient correlated well with protein hydrophobicity at low and high concentrations of NaCl (r = 0.81 and 0.93, respectively). The PEG/citrate system also had a higher hydrophobic resolution than other systems to exploit differences in the protein's hydrophobicity. The surface charge and charge density of the proteins was determined over a range of pH (3-9) by electrophoretic titration curves; PEG/salt systems did not discriminate well between proteins of different charge or charge density. In the absence of NaCl, K decreased slightly with increased positive charge. At high NaCl concentration, K increased as a function of positive charge. This suggested that the PEG-rich top phase became more negative as the concentration of NaCl in the systems increased and, therefore, attracted the positively charged proteins. The effect of charge was more important in PEG/dextran systems at low concentrations of NaCl. In the PEG/dextran systems at lower concentration of NaCl, molecular weight appeared to be the prime determinant of partition, whereas no clear effect of molecular weight could be found in PEG/salt systems.     

Effect of molecular crowding on DNA polymerase activity

Live cells contain high concentrations of macromolecules, but almost all experimental biochemical data have been generated from dilute solutions that do not reflect conditions in vivo. To understand biomolecular behavior in vivo, properties studied in vitro are extrapolated to conditions in vivo; however, the molecular conditions within live cells are inherently crowded. The present study investigates the effect of molecular crowding on DNA polymerase activity using polyethylene glycol PEG of various molecular weights as a crowding agent. Polymerase activity assays under various conditions demonstrated that the activities of T7 and Taq DNA polymerases depend on the molecular weight and concentration of the crowding agent. Furthermore, equilibrium and kinetic analyses demonstrated that the binding affinity and catalytic activity of the polymerase increase and decrease, respectively, with increasing PEG concentrations. Based on quantitative parameters of the polymerase reactions, we improved the efficiency of PCR amplification under conditions of molecular crowding. These results suggest that quantitative measurements of biomolecular structure and function are useful for understanding the behavior of biomolecules in vivo and for biotechnology applications in vitro.     

Polyethylene glycol-induced mammalian cell hybridization: effect of polyethylene glycol molecular weight and concentration.

The effects of polyethylene glycol (PEG) molecular weight and concentration on mammalian cell hybridization were studied. The peak hybridization-inducing activity with all grades of PEG from 400-6000 was found to occur in the concentration range of 50-55%. However, changes in concentration were seen to have different quantitative effects with different grades of PEG. For monolayer fusions, PEG 1000 at 50% seems to be the optimal combination of PEG molecular weight and concentration, in terms of both efficiency of hybridization and relative insensitivity to dilution effects.     

Aqueous two-phase extraction for protein recovery from corn extracts

Corn has been used as an expression host for several recombinant proteins with potential for large-scale production. Cost-effective downstream initial recovery, separation and concentration remain a challenge. Aqueous two-phase (ATP) partitioning has been used to recover and concentrate proteins from fermentation broths and offers advantages for integration of those steps with biomass removal. To examine the applicability of ATP partitioning to recombinant protein purification from corn endosperm and germ, ATP system parameters including poly(ethylene glycol) (PEG) molecular weight (MW), phase-forming salt, tie line length (TLL), and pH were manipulated to control partitioning of extracted native proteins from each fraction. Moderate PEG MW, reduction of phase ratio, and added NaCl effected complete recovery of the hydrophobic model protein lysozyme in the top phase with ca. 5x enrichment and illustrates a favorable match of recombinant protein characteristics, expression host, and separation method. Furthermore, integration of protein extraction with the partitioning reduced the load of contaminating host proteins relative to the more traditional separate steps of extraction followed by partitioning. Performance of the integrated partitioning was hindered by endosperm solids loading, whereas for germ, which has ca. 35x higher aqueous soluble protein, the limit was protein solubility. For more hydrophilic model proteins (the model being cytochrome c), effective separation required further reduction of PEG MW to effect more partitioning of host proteins to the top phase and enrichment of the model protein in the lower phase. The combination of PEG MW of 1450 with 8.5 wt.% NaCl addition (Na(2)SO(4) as the phase-forming salt) provided for complete recovery of cytochrome c in the lower phase with enrichment of 9x (germ) and 5x (endosperm). As a result of lower-phase recovery, the advantage of simultaneous removal of solids is lost. The lower solubility of native endosperm proteins results in higher purity for the same enrichment.     

Protein isolation by solution-controlled gel sorption.

Illustrated are the principles for isolating proteins from solution by sorption into a polymer gel phase, driven by the addition of a water-soluble polymer to the protein solution. The separation is shown to be analogous to conventional two-phase aqueous extraction. However, the use of a gel phase rather than a solution for absorbing the protein makes separation of the protein from the polymer and the recycling of the gel phase much simpler. The model system used was linear poly(ethylene glycol) (PEG) and dextran gel. Increasing the molecular weight and concentration of the PEG favored sorption by the gel of ovalbumin, bovine serum albumin, cytochrome c, and hemoglobin. The proteins could be quantitatively recovered by immersing the gel in PEG-free solution.     

Protein partitioning at the isoelectric point: influence of polymer molecular weight and concentration and protein size

We report the partition coefficient, K(p') at the isoelectric point of lysozyme, chymotrypsinogen A, albumin, transferrin, and catalase in 64 different polyethylene(PEG)/ dextran(Dx)/water systems. We study the trends of the partition coefficient with protein type, polymer concentration, and polymer molecular weight. We find that the partition coefficient decreases with increasing tie line length for lysozyme, albumin, transferrin, and catalase for which K(p) is less than 1, but increases for chymotrysinogen for which K(p) is larger than 1. The effect of the tie line length on the partition coefficient is larger for the large proteins than for the small proteins. The partition coefficient decreases with increasing protein molecular weight except for lysozyme suggesting that lysozyme is present as a dimer or a trimer. The partition coefficient decreases with increasing PEG molecular weight, but the magnitude of the increase is larger for the smaller PEG molecular eights and tends to level of at high PEG molecular weight. The partition coefficient increases with increasing dextran (Dx) molecular weight for chymotrypsinogen but decreases for catalase. The partition coefficients of lysozyme, albumin, and transferrin increase with increasing Dx molecular weight from Dx 10(4) to Dx 1.1 x 10(5) and then slightly decrease from Dx 1.1 x 10(5) to Dx 5 x 10(5). The experimental results are analyzed using a statistical thermodynamics model. The experimental results are analyzed using a statistical thermodynamics model. The experiments suggest that protein partitioning at the isoelectric point in aqueous two-phase systems is strongly related to the size of the proteins and polymers. Finally, the impossibility of obtaining data completely independent of polymer concentration is emphasized.     

Model‐Based Investigation on the Mass Transfer and Adsorption Mechanisms of Mono‐Pegylated Lysozyme in Ion‐Exchange Chromatography

Recent studies highlighted the potential of PEGylated proteins to improve stabilities and pharmacokinetics of protein drugs. Ion‐exchange chromatography (IEX) is among the most frequently used purification methods for PEGylated proteins. However, the underlying physical mechanisms allowing for a separation of different PEGamers (proteins with a varying number of attached PEG molecules) are not yet fully understood. In this work, mechanistic chromatography modeling is applied to gain a deeper understanding of the mass transfer and adsorption/desorption mechanisms of mono‐PEGylated proteins in IEX. Using a combination of the general rate model (GRM) and the steric mass action (SMA) isotherm, simulation results in good agreement with the experimental data are achieved. During linear gradient elution of proteins attached with PEG of different molecular weight, similar peak heights, and peak shapes at constant gradient length are observed. A superimposed effect of increased desorption rate and reduced diffusion rate as a function of the hydrodynamic radius of PEGylated proteins is identified to be the reason of this anomaly. That is why the concept of the diffusion‐desorption‐compensation effect is proposed. In addition to the altered elution orders, PEGylation results in a considerable decrease of maximum binding capacity. By using the SMA model in a kinetic formulation, the adsorption behavior of PEGylated proteins in the highly concentrated state is described mechanistically. An exponential increase in the steric hindrance effect with increasing PEG molecular weight is observed. This suggests the formation of multiple PEG layers in the interstitial space between bound proteins and an associated shielding of ligands on the adsorber surface to be the cause of the reduced maximum binding capacity. The presented in silico approach thus complements the hitherto proposed theories on the binding mechanisms of PEGylated proteins in IEX.     

Size fractionation of double-stranded DNA by precipitation with polyethylene glycol

We show that DNA molecules of differing molecular mass are separable by selective precipitation with polyethylene glycol (PEG+.. Higher molecular mass DNA precipitates at lower PEG concentrations than lower molecular mass DNA. Double-stranded DNA can be fractionated at least in the range of 3 times 10-7 to 1 times 10-5 daltons. The effects on PEG concentration, sodium chloride concentration, DNA concentration, pH, divalent ions, precipitation time, and centrifugal force have been determined. These studies show PEG precipitation offers a size fractionation method for DNA which is convenient, of high capacity, and applicable over a wide range of conditions. However, resolution is not high and separation of two species approaches 100% only if they differ in molecular mass by at least a factor of two.     

Partitioning features of bovine trypsin and alpha-chymotrypsin in polyethyleneglycol-sodium citrate aqueous two-phase systems

The partitioning of bovine trypsin and alpha-chymotrypsin--proteases of similar physico-chemical properties--in different polyethyleneglycol/sodium citrate aqueous two-phase systems was investigated. The effect of different factors such as polyethyleneglycol molecular weight, pH, tie line length, temperature and the presence of an inorganic salt on the protein partition coefficient were analysed. Both a decrease in PEG molecular weight and an increase in pH led to a higher partition coefficient for both enzymes. Aqueous two-phase systems formed by PEG of molecular weight 3350 and citrate pH 5.2 showed the best separation capability which was enhanced in presence of sodium chloride 3%. The transfer of both proteins to the top phase was associated with negative enthalpic and entropic changes.     

Mechanisms of phase behaviour and protein partitioning in detergent/polymer aqueous two-phase systems for purification of integral membrane proteins

Detergent/polymer aqueous two-phase systems are studied as a fast, mild and efficient general separation method for isolation of labile integral membrane proteins. Mechanisms for phase behaviour and protein partitioning of both membrane-bound and hydrophilic proteins have been examined in a large number of detergent/polymer aqueous two-phase systems. Non-ionic detergents such as the Triton series (polyoxyethylene alkyl phenols), alkyl polyoxyethylene ethers (C(m)EO(n)), Tween series (polyoxyethylene sorbitol esters) and alkylglucosides form aqueous two-phase systems in mixtures with hydrophilic polymers, such as PEG or dextran, at low and moderate temperatures. Phase diagrams for these mixtures are shown and phase behaviour is discussed from a thermodynamic model. Membrane proteins, such as bacteriorhodopsin and cholesterol oxidase, were partitioned strongly to the micelle phase, while hydrophilic proteins, BSA and lysozyme, were partitioned to the polymer phase. The partitioning of membrane protein is mainly determined by non-specific hydrophobic interactions between detergent and membrane protein. An increased partitioning of membrane proteins to the micelle phase was found with an increased detergent concentration difference between the phases, lower polymer molecular weight and increased micelle size. Partitioning of hydrophilic proteins is mainly related to excluded volume effects, i.e. increased phase component size made the hydrophilic proteins partition more to the opposite phase. Addition of ionic detergent to the system changed the partitioning of membrane proteins slightly, but had a strong effect on hydrophilic proteins, and can be used for enhanced separation between hydrophilic proteins and membrane protein.     

Enhancement effect of polyethylene glycol on enzymatic synthesis of cephalexin

In an enzymatic synthesis of cephalexin (CEX) using an acylase from Xanthomonas citri, the effect of polyethylene glycol (PEG) on the synthetic reaction of 7-amino-3-deacetoxycephalosporanic acid (7-ADCA) and D-alpha-phenyl-glycine methyl ester (PGM) to CEX was investigated. The addition of PEG (MW 300-20,000) increased the yield significantly. This yield enhancement effect tended to increase with the increasing molecular weight of PEG. Addition of PEG to the reaction system did not affect both the CEX and PGM hydrolytic reactions. The PEG added to the reaction medium used in these experiments did not depress the water activity significantly, and the product yield improvement could not be explained by the activity alone. The PEG stabilized the enzyme activity to some extent, but this stabilizing effect was only partially attributable to the yield enhancement of CEX. The enhancing effect of PEG on the synthetic yield increased with the increasing PEG molecular weight or the length of the poly(oxy-1,2-ethanediyl) chain, which increases the hydrophobicity of PEG. This finding consequently has led to the conclusion that the PEG structure renders the affinity between enzyme and 7-ADCA, which is a hydrophobic substrate. The microenvironmental hydrophobicity of PEG and its interaction with the hydrophobic substrate was found to be the main reason for the improvement of the CEX yield. In fact, the Michaelis-Menten kinetic constant for 7-ADCA, K(7-ADCA) in the presence of PEG was smaller than that in the control system (without PEG addition). (c) 1993 John Wiley & Sons, Inc.     

Protein sorption and recovery by hydrogels using principles of aqueous two-phase extraction

Use of the thermodynamic principles of aqueous two-phase extraction (ATPE) to drive protein into a crosslinked gel is developed as a protein isolation and separation technique, and as a protein loading technique for drug delivery applications. A PEG/dextran gel system was chosen as a model system because PEG/dextran systems are widely used in aqueous two-phase extraction and dextran gels (Sephadex(R)) are common chromatographic media. The effects of polymer concentrations and molecular weights, salts, and pH on the partitioning of ovalbumin matched ATPE heuristics and data trends. Gel partition coefficients (Cgel/Csolution) increased with increasing PEG molecular weight and concentration and decreasing dextran concentration (increased gel swelling). The addition of PEG to the buffer solution yielded partition coefficients more than an order of magnitude greater than those obtained in systems with buffer alone, or added salt. A combined salt/PEG system yielded an additional order of magnitude increase. For example, when ovalbumin solution (2.3 mg/mL) was equilibrated with Sephadex(R) G-50 at pH 6.75, the partition coefficients were 0.13 in buffer, 0.11 in buffer with 0.22M KI, 2.3 in 12 wt% PEG-10,000 and 32.0 in 12 wt% PEG-10, 000 with 0.22M KI. The effect of anions and cations as well as ionic strength and pH on the partitioning of ovalbumin also matched ATPE heuristics. Using the heuristics established above, partition coefficients as high as 80 for bovine serum albumin and protein recoveries over 90% were achieved. In addition, the wide range of partition coefficients that were obtained for different proteins suggests the potential of the technique for separating proteins. Also, ovalbumin sorption capacities in dextran were as high as 450 mg/g dry polymer, and the sorption isotherms were linear over a broad protein concentration range.     

The effects of proteins secreted by fibroblasts from patients with cystic fibrosis on hamster tracheal explants

Summary Hamster tracheal explants have been used to assay for mucosecretory activity in media taken from cultures of fibroblasts isolated from patients with cystic fibrosis (CF). Cystic fibrosis and normal sera were first used to establish optimal conditions for mucus release in the hamster tracheal ring assay. Unless protein levels were maintained at 5% serum concentration or greater there was loss of cilia, nonspecific mucus accumulation, and extensive epithelial damage to the luminal surface. Likewise, it was shown that exposure of the explants to unconcentrated conditioned media from CF (GM 770, 768, 1348, 142) or normal (GM 3349, 38) cultured fibroblasts for 1, 6, or 12 h resulted in the same type of damage and this was due to low protein levels. When the protein concentration of the conditioned media was increased with fetal bovine serum, the morphological integrity of the explants was maintained, demonstrating that there was no apparent difference between CF and normal fibroblast-secreted proteins in ability to induce mucus release. The ciliary inhibitory capacity of CF serum-derived or fibroblast-derived factor had been reported to require IgG for activity. However, addition of IgG to high molecular weight (VoP10) or low molecular weight (VeP10) secreted proteins had no apparent effect on stimulating secretion. In conclusion, it is possible that CF fibroblasts do not secrete a protein that has the mucostimulatory effect and thus these cells may not be suitable for studying the CF-related activity.     

Detection and molecular weight determination of polyethylene glycol-modified hirudin by staining after sodium dodecyl sulfate-polyacrylamide gel electrophoresis.

This article describes a general method for detecting pegylated proteins directly after SDS-PAGE. The proteins to which polyethylene glycol (PEG) molecules are attached are stained with a barium iodide solution. The staining is based on the formation of a barium iodide complex with PEG. The described method combines a specific staining of PEG molecules with the high resolution of the SDS-PAGE method. It is shown that pegylated protein is detectable on SDS-PAGE as well as on IEF at concentrations that are not detectable by Coomassie protein staining. This paper also describes the determination of the molecular weight of pegylated hirudin by calibrating SDS-PAGE with polyethylene glycol of different molecular weight. Under the conditions used, PEG showed linear mobility during electrophoresis. However, the use of nonpegylated proteins as standards resulted in incorrect molecular weight values due to the lower mobility of the pegylated protein during electrophoresis. The method described might reflect a general method for determining molecular weight of pegylated proteins.     

Spectroscopic characterization of polyethyleneglycol modified superoxide dismutase: 1H NMR studies on its Cu2Co2 derivative

Spectroscopic methods have been employed in order to understand the molecular basis of the decrease in enzymatic activity of the antiinflammatory enzyme copper-zinc superoxide dismutase (SOD) following the covalent binding of polyethyleneglycol (PEG) chains to the protein amino-groups. The PEG modification is a general method recently proposed to improve the therapeutic index of enzymes. 1H NMR spectra on the cobalt substituted PEG-modified SOD, Cu2Co2-PEG-SOD, have been recorded. The signals are quite broad with respect to the unmodified enzyme. This has been interpreted on the basis of the effect of molecular weight on the linewidth. The analysis has shown that the histidine hydrogens involved in metal binding at the enzyme active site are the same in both native and PEG-modified SOD. Similarly, circular dichroism and absorption spectra indicate that the overall conformation of the metal clusters is not perturbed upon modification. On the other hand, azide titration shows that the affinity constant of N-3 for SOD is largely reduced upon PEG modification (K = 154 M-1 and 75 M-1 for the native and modified SOD, respectively). These results indicate that the decrease in enzymatic activity upon surface modification with PEG is not caused by a perturbation of the active site geometry, but to a decrease in the channeling of the O2- ion towards the enzyme active site.     

Interaction of poly(ethylene-glycols) with air-water interfaces and lipid monolayers: investigations on surface pressure and surface potential.

We have characterized the surface activity of different-sized poly(ethylene-glycols) (PEG; M(r) 200-100,000 Da) in the presence or absence of lipid monolayers and over a wide range of bulk PEG concentrations (10(-8)-10% w/v). Measurements of the surface potential and surface pressure demonstrate that PEGs interact with the air-water and lipid-water interfaces. Without lipid, PEG added either to the subphase or to the air-water interface forms relatively stable monolayers. Except for very low molecular weight polymers (PEGs < 1000 Da), low concentrations of PEG in the subphase (between 10(-5) and 10(-4)% w/v) increase the surface potential from zero (with respect to the potential of a pure air-water interface) to a plateau value of approximately 440 mV. At much higher polymer concentrations, > 10(-1)% (w/v), depending on the molecular weight of the PEG and corresponding to the concentration at which the polymers in solution are likely to overlap, the surface potential decreases. High concentrations of PEG in the subphase cause a similar decrease in the surface potential of densely packed lipid monolayers spread from either diphytanoyl phosphatidylcholine (DPhPC), dipalmitoyl phosphatidylcholine (DPPC), or dioleoyl phosphatidylserine (DOPS). Adding PEG as a monolayer at the air-water interface also affects the surface activity of DPhPC or DPPC monolayers. At low lipid concentration, the surface pressure and potential are determined by the polymer. For intermediate lipid concentrations, the surface pressure-area and surface potential-area isotherms show that the effects due to lipid and PEG are not always additive and that the polymer's effect is distinct for the two lipids. When PEG-lipid-mixed monolayers are compressed to surface pressures greater than the collapse pressure for a PEG monolayer, the surface pressure-area and surface potential-area isotherms approach that of the lipid alone, suggesting that for this experimental condition PEG is expelled from the interface.