Mechanisms of phase behaviour and protein partitioning in detergent/polymer aqueous two-phase systems for purification of integral membrane proteins1

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 (CmEOn), 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

Interaction between tryptophan residues and hydrophobically modified dextran - Effect on partitioning of peptides and proteins in aqueous two-phase systems

Hydrophobically modified dextrans, benzoyl dextran and valeryl dextran, have been used to study the interactions between tryptophan residues and benzoyl or valeryl groups by partitioning of tryptophan, tryptophan-tryptophan, (tryptophan)3, poly(lysine, tryptophan), β-galactosidase and lysozyme in polymer aqueous two-phase systems. The two-phase systems used were polyethylene glycol (PEG)-benzoyl dextran, PEG-valeryl dextran, dextran-benzoyl dextran and dextran-valeryl dextran. Interaction between tryptophan residues and benzoyl or valeryl groups was observed by partitioning of tryptophan containing compounds to the phase containing hydrophobically modified dextran. At a certain phase composition the interactions were increased with increasing number of tryptophan per molecule. In a PEG-dextran system the partitioning of tryptophan peptides to the PEG phase was increased with increased number of tryptophan. In a PEG-benzoyl dextran system the opposite effect was obtained. At similar conditions benzoyl groups showed stronger interactions with tryptophans compared to valeryl groups. The partition coefficient of salts (sodium phosphate, NaCl, NaI and NaClO4) was determined in PEG-benzoyl dextran and PEG-valeryl dextran aqueous two-phase systems. The effect of addition of these salts on partitioning of poly(lysine, tryptophan), β-galactosidase and lysozyme was studied. Salt effects on partitioning could be explained by the relative affinities of the ions for the polymers in the system. Charged molecules containing tryptophan were to an increasing degree partitioned to the phase for which the counterions had highest affinity. Strong effects on the partitioning of positively charged poly(lysine, tryptophan) and lysozyme were obtained with the ions I- and ClO4-

Peptide fusion tags with tryptophan and charged residues for control of protein partitioning in PEG-potassium phosphate aqueous two-phase systems

A partition study with peptides and recombinant proteins in poly(ethylene glycol)4000-potassium phosphate aqueous two-phase systems has been performed. The aim was to study to what extent the insertion of charged residues could affect protein partition in addition to the already observed effects of tryptophan residues. The model proteins used are based on a staphylococcal protein A derivative, Z, and modified by the insertion of peptide tags close to the C-terminus. The tags differed with respect to their content of both Trp, negatively (Asp) and positively charged (Lys) amino acid residues. The same partitioning trends were observed for the peptides and fusion proteins. The effect of Trp residues was to direct the partitioning towards the PEG phase. The insertion of two negatively charged (Asp) residues into a Trp_4-tag enhanced the partition towards the PEG phase even more. The introduction of positively charged (Lys) residues in addition to Trp residues, on the other hand, pulled the peptide or protein towards the potassium phosphate phase. The partitioning of peptides gave a good qualitative picture of the effect of the peptide on partitioning when fused to the protein. The efficiencies of the tags were calculated based on partitioning of tags and fusion proteins, and tag efficiencies generally varied between 60 and 85%

Ion-exchange chromatographic purification and quantitative analysis of Trichoderma reesei cellulases cellobiohydrolase I, II and endoglucanase II by fast protein liquid chromatography

Trichoderma cellulases appear in several isoforms which makes their purification and analysis difficult. We used fast protein liquid chromatography (FPLC) to purify three major cellulases and to quantitate these enzymes in reconstituted mixtures during cellulose hydrolysis studies (in lack of specific substrates and because of the synergism between the enzymes such analysis is very difficult, if at all possible, with conventional activity measurements). For the analysis methods linear calibration was achieved from 10-15 pmol to 0.5-1 nmol (from 0.5-0.8 to 27-64 μg) for the different enzymes. Due to the high resolution chromatographic media used, our purification methods are simpler and quicker than the usual protocols for cellulase purification. Several isoforms of cellobiohydrolase (CBH) I were purified. The isoforms had different isoelectric points (pI) but their catalytic and adsorption properties were similar. A remarkable feature of CBH I and endoglucanase (EG) II was that their electrophoretically pure preparations gave double peaks during ion-exchange chromatography in certain pH intervals where the two peaks (probably representing two conformations) were transformed into each other by changing pH. This behaviour of cellulases has never been reported before and further explains the difficulties in cellulase purification

Partitioning in Aqueous Polymer 2-phase Systems .1. Modeling of Affinity Partition

Various factors influencing affinity partitioning in aqueous polymer two-phase systems are studied by model calculations using a self-consistent mean-field lattice theory. The latter is an extension of the theory of Scheutjens and Fleer, adapted to take affinity ligand binding into account. The dependence of partition coefficients on ligand concentration is studied for different binding strengths, polymer lengths, and polymer-protein interactions, and the effects of the ligand being attached at different positions of the polymer chain are investigated. The mechanism of the affinity partitioning is discussed. In particular, at saturation the enhanced partitioning by the presence of the ligand substituted polymers should be regarded as an expulsion from the minority phase rather than an attraction to the majority phase. Moreover, the results are put into relation with the multiple-equilibriascheme that has frequently been used to analyse affinity partition data, and comparisons with experimental findings are made. Some important principles for application of affinity partitioning concluded from the modelling results are: i) A higher selectivity can be expected if a polymer which has in general repulsive interactions with proteins is chosen as ligand-carrying polymer, ii) a higher Delta(lnK)(max) is expected for a longer ligand-carrying polymer as compared to a shorter one. With increased polymer length higher ligand concentration is however needed to reach the plateau value. iii) the ligand bound to the end of the polymer gives a higher Delta(lnK)(max) than when bound to the middle of the chain. iv) having both polymer ends carrying ligand should actually lead to a decrease in the Delta(lnK)(max). However, at quite low ligand concentrations the doubly substituted ligand-carrier should be more effective

A model explaining declining rate in hydrolysis of lignocellulose substrates with cellobiohydrolase I (cel7A) and endoglucanase I (cel7B) of Trichoderma reesei

It is commonly observed that the rate of enzymatic hydrolysis of solid cellulose substrates declines markedly with time. In this work the mechanism behind the rate reduction was investigated using two dominant cellulases of Trichoderma reesei: exoglucanase Cel7A (formerly known as CBHI) and endoglucanase Cel7B (formerly EGI). Hydrolysis of steam-pretreated spruce (SPS) was performed with Cel7A and Cel7B alone, and in reconstituted mixtures. Throughout the 48-h hydrolysis, soluble products, hydrolysis rates, and enzyme adsorption to the substrate were measured. The hydrolysis rate for both enzymes decreases rapidly with hydrolysis time. Both enzymes adsorbed rapidly to the substrate during hydrolysis. Cel7A and Cel7B cooperate synergistically, and synergism was approximately constant during the SPS hydrolysis. Thermal instability of the enzymes and product inhibition was not the main cause of reduced hydrolysis rates. Adding fresh substrate to substrate previously hydrolyzed for 24 h with Cel7A slightly increased the hydrolysis of SPS; however, the rate increased even more by adding fresh Cel7A. This suggests that enzymes become inactivated while adsorbed to the substrate and that unproductive binding is the main cause of hydrolysis rate reduction. The strongest increase in hydrolysis rate was achieved by adding Cel7B. An improved model is proposed that extends the standard endo-exo synergy model and explains the rapid decrease in hydrolysis rate. It appears that the processive action of Cel7A becomes hindered by obstacles in the lignocellulose substrate. Obstacles created by disordered cellulose chains can be removed by the endo activity of Cel7B, which explains some of the observed synergism between Cel7A and Cel7B. The improved model is supported by adsorption studies during hydrolysis

Isotherms for adsorption of cellobiohydrolase I and II from Trichoderma reesei on microcrystalline cellulose

Adsorption to microcrystalline cellulose (Avicel) of pure cellobiohydrolase I and II (CBH I and CBH II) from Trichoderma reesei has been studied. Adsorption isotherms of the enzymes were measured at 4 degrees C using CBH I and CBH II alone and in reconstituted equimolar mixtures. Several models (Langmuir, Freundlich, Temkin, Jovanovic) were tested to describe the experimental adsorption isotherms. The isotherms did not follow the basic (one site) Langmuir equation that has often been used to describe adsorption isotherms of cellulases; correlation coefficients (R-2) were only 0.926 and 0.947, for CBH I and II, respectively. The experimental isotherms were best described by a model of Langmuir type with two adsorption sites and by a combined Langmuir-Freundlich model (analogous to the Hill equation); using these models the correlation coefficients were in most cases higher than 0.995. Apparent binding parameters derived from the two sites Langmuir model indicated stronger binding of CBH II compared to CBH I; the distribution coefficients were 20.7 and 3.7 L/g for the two enzymes, respectively. The binding capacity, on the other hand, was higher for CBH I, 1.0 mu mol (67 mg) per gram Avicel, compared to 0.57 mu mol/g (30 mg/g) for CBH II. The isotherms when analyzed with the combined Langmuir-Freundlich model indicated presence of unequal binding sites on cellulose and/or negative cooperativity in the binding of the enzyme molecules