Exploitation of specific properties of trifluoroethanol for extraction and separation of membrane proteins

Hydrophobic proteins are difficult to analyze by two-dimensional electrophoresis (2-DE) because of their intrinsic tendency to self-aggregate during the first dimension (isoelectric focusing, IEF) or the equilibration steps. This aggregation renders their redissolution for the second dimension uncertain and results in the reduction of the number and intensity of protein spots, and in undesirable vertical and horizontal streaks across gels. Trifluoroethanol (TFE) is traditionally used at high concentration to solubilize peptides and proteins for NMR studies. Depending upon its concentration, TFE strongly affects the three-dimensional structure of proteins. We report here a phase separation system based on TFE/CHCl(3), which is able to extract a number of intrinsic membrane proteins. The addition of TFE in the in-gel sample rehydration buffer to improve membrane protein IEF separation is also presented. The procedure using urea, thiourea, and sulfobetaine as chaotropic agents was modified by the addition of TFE and removing of sulfobetaine at an optimized concentration in the solubilization medium used for the first dimension. When using membrane fractions isolated from Escherichia coli, the intensity and the number of spots detected from 2-DE gels that used TFE in the solubilization medium were significantly increased. The majority of the proteins identified using peptide mass fingerprinting and tandem mass spectrometry (MS/MS) were intrinsic membrane proteins, proteins of beta barrel structure or transmembrane proteins.     

Butanol recovery from fermentations by liquid-liquid extraction and membrane solvent extraction

Extraction can successfully be used for in-situ alcohol recovery in butanol fermentations to increase the substrate conversion. An advantage of extraction over other recovery methods may be the high capacity of the solvent and the high selectivity of the alcohol/water separation. Extraction, however, is a comprehensive operation, and the design of an extraction apparatus can be complex. The aim of this study is to assess the practical applicability of liquid-liquid extraction and membrane solvent extraction in butanol fermentations. In this view various aspects of extraction processes were investigated.Thirty-six chemicals were tested for the distribution coefficient for butanol, the selectivity of alcohol/water separation and the toxicity towards Clostridia. Convenient extractants were found in the group of esters with high molar mass.Liquid-liquid extraction was carried out in a stirred fermenter and a spray column. The formation of emulsions and the fouling of the solvent in a fermentation broth causes problems with the operation of this type of equipment. With membrane solvent extraction, in which the solvent is separated from the broth by a membrane, a dispersion-free extraction is possible, leading to an easy operation of the equipment. In this case the mass transfer in the membrane becomes important.With membrane solvent extraction the development of a process is emphasized in which the extraction characteristics of the solvent are combined with the property of silicone rubber membranes to separate butanol from water. In the case of apolar solvents with a high molar mass, the characteristics of the membrane process are determined completely by the solvent. In the case of polar solvents (e.g. ethylene glycol), the permselectivity of the membrane can profitably be used. This concept leads to a novel type of extraction process in which alcohol is extracted with a water-soluble solvent via a hydrophobic semipermeable membrane. This extraction process has been investigated for the recovery of butanol and ethanol from water. A major drawback in all processes with membrane solvent extraction was the permeation of part of the solvent to the aqueous phase.The extraction processes were coupled to batch, fed batch and continuous butanol fermentations to affirm the applicability of the recovery techniques in the actual process. In the batch and fed batch fermentations a three-fold increase in the substrate consumption could be achieved, in the continuous fermentation about 30% increase.     

Polymer-induced phase separation in aqueous micellar solutions of octyl-beta-D-thioglucoside for extraction of membrane proteins

A water-soluble polymer such as polyethylene glycol (PEG), Dextran T-500 (Dx), or diethylaminoethyl-Dextran (DEAE-Dx) induced aqueous micellar solutions of octyl-beta-D-thioglucoside (OTG) to phase separation at 0 degrees C. One of the two phases thus formed is a surfactant-depleted aqueous solution (aqueous phase) of a water-soluble polymer and the other a concentrated OTG solution (surfactant-rich phase). In a combination of OTG with PEG or Dx, cytochrome P450 (P450) and cytochrome b(5) (b(5)) were well extracted into the surfactant-rich phase. The extraction yield of P450 was slightly greater than that of b(5). In contrast to PEG and Dx, DEAE-Dx markedly reduced the extraction of b(5), while that of P450 remained almost unchanged. DEAE-Dx served the dual functions of inducing the phase separation and preventing the extraction of b(5) into the surfactant-rich phase. This depressed extraction of b(5) was reversed by the addition of potassium phosphate. DEAE-Dx and potassium phosphate proved effective in controlling the extractability of b(5). The polymer-induced phase separation provides a new basis for highly efficient extraction of membrane proteins under mild conditions that should be acceptable for thermolabile membrane proteins under physiological conditions. (c) 1997 John Wiley & Sons, Inc. Biotechnol Bioeng 56: 311-318, 1997.     

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.     

Evaluation of chloroform/methanol extraction to facilitate the study of membrane proteins of non-model plants

Membrane proteins are of great interest to plant physiologists because of their important function in many physiological processes. However, their study is hampered by their low abundance and poor solubility in aqueous buffers. Proteomics studies of non-model plants are generally restricted to gel-based methods. Unfortunately, all gel-based techniques for membrane proteomics lack resolving power. Therefore, a very stringent enrichment method is needed before protein separation. In this study, protein extraction in a mixture of chloroform and methanol in combination with gel electrophoresis is evaluated as a method to study membrane proteins in non-model plants. Benefits as well as disadvantages of the method are discussed. To demonstrate the pitfalls of working with non-model plants and to give a proof of principle, the method was first applied to whole leaves of the model plant Arabidopsis. Subsequently, a comparison with proteins extracted from leaves of the non-model plant, banana, was made. To estimate the tissue and organelle specificity of the method, it was also applied on banana meristems. Abundant membrane or lipid-associated proteins could be identified in both tissues, with the leaf extract yielding a higher number of membrane proteins.     

Novel polymer-polymer conjugates for recovery of lactic acid by aqueous two-phase extraction

A new family of polymer conjugates is proposed to overcome constraints in the applicability of aqueous two-phase systems for the recovery of lactic acid. Polyethylene glycol-polyethylenimine (PEI) conjugates and ethylene oxide propylene oxide-PEI (EOPO-PEI) conjugates were synthesized. Aqueous two-phase systems were generated when the conjugates were mixed with fractionated dextran or crude hydrolyzed starch. With 2% phosphate buffer in the systems, phase diagrams with critical points of 3.9% EOPO-PEI-3.8% dextran (DEX) and 3.5% EOPO-PEI-7.9% crude starch were obtained. The phase separation temperature of 10% EOPO-PEI solutions titrated with lactic acid to pH 6 was 35 degrees C at 5% phosphate, and increased linearly to 63 degrees C at 2% phosphate. Lactic acid partitioned to the top conjugate-rich phase of the new aqueous two-phase systems. In particular, the lactic acid partition coefficient was 2.1 in 10% EOPO-PEI-8% DEX systems containing 2% phosphate. In the same systems, the partitioning of the lactic acid bacterium, Lactococcus lactis subsp. lactis, was 0.45. The partitioning of propionic, succinic, and citric acids was also determined in the new aqueous two-phase systems.     

Large-scale separation and production of engineered proteins,designed for facilitated recovery in detergent-based aqueous two-phase extraction systems

The feasibility and scalability of extraction in detergent-based aqueous two-phase systems for the separation of proteins from culture broth is demonstrated. At the same time the large-scale production of a fusion protein and the influence of cultivation scale on the efficiency of separation were investigated. An amphiphilic fusion protein EGIcore-HFBI was chosen, consisting of the catalytic core of the cellulase endoglucanase I and the small protein hydrophobin I, expressed homologously in Trichoderma reesei. Using the technical nonionic detergent Agrimul NRE 1205 the separation was successfully scaled up to 1200 l. No differences in yield or in partition coefficient were observed at 10 ml and 1200 l scale. Changes in the fermentation temperature and scale, however, can influence the properties of the protein and thus alter partition coefficient and yield. The decreased separation efficiency appears to be correlated with changes in glycosylation at lower cultivation temperatures.     

Scale-up of recombinant cutinase recovery by whole broth extraction with PEG-phosphate aqueous two-phase

A whole broth extraction using an aqueous two-phase system (ATPS) composed by 5% (w/w) PEG 3350 and 15% (w/w) phosphate was used for the scale-up extraction and isolation of a recombinant Fusarium solani pisi cutinase, an extracellular mutant enzyme expressed in Saccharomyces cerevisiae, containing a fusion peptide (WP)₄. The experiments were carried out at three different scales (10 ml, 1 l and 30 l). Mixing time and stirrer speed were evaluated at lab scale (1 l) with two different system compositions. Stirrer speed between 400 and 800 rpm and mixing time between 2 and 5 min led to the highest recoveries of cutinase. In all cases, inclusive of pilot scale (30 l), the equilibrium was reached after a few minutes. The performance of ATPS was reproducible within the scale range of 0.010–30 l and provided a standard deviation of the yield lower than 8%, leading to (i) a partition coefficient over 50, (ii) a yield over 95% and (iii) a concentration factor over 5. The fusion of the peptide (WP)₄ to the cutinase protein enabled a 400 increase of the partition coefficient relative to the wild-type strain.     

Ion selectivity of aqueous leaks induced in the erythrocyte membrane by crosslinking of membrane proteins

The aqueous leak induced in the human erythrocyte membrane by crosslinking of spectrin via disulfide bridges formed in the presence of diamide (Deuticke, B., Poser, B., Lütkemeier, P. and Haest, C.W.M. (1983) Biochim. Biophys. Acta 731, 196-210) was further characterized with respect to its ion selectivity by means of (a) measurements of cell volume changes or hemolysis, (b) determination of membrane potentials and (c) analysis of potential-driven ion fluxes. The leak turned out to be slightly cation-selective (PK:PCl approximately equal to 4:1). It discriminates mono- from divalent ions (PNa:PMg greater than 100:1, PCl:PSO4 greater than 10:1) and to a much lesser extent monovalent ions among each other. The selectivities for monovalent ions follow the sequence of free solution mobilities, increasing in the order Li+ less than or equal to Na+ less than K+ less than or equal to Rb+ less than Cs+ and F- less than Cl- less than Br- less than I-. Polyatomic anions also fit into that order. Quantitatively, the ratios of permeabilities of the leak are larger than those of the ion mobilities in free solution. The ion permeability of the leak is concentration-independent up to at least 150 mM. The ion milieu, however, has marked effects on leak permeability, most pronounced for chaotropic ions (guanidinium, nitrate, thiocyanate), which increase leak fluxes of charged and uncharged solutes. The results support the view that, besides geometric constraints, weak coulombic or dipolar interactions between penetrating ions and structural elements of the leak determine permselectivity.     

Differential dynamics of membrane proteins in yeast

Lateral diffusion of lipids and proteins in yeast plasma membranes has been reported to be anomalously slow, and implicated as a possible reason for polarization in yeast. In order to gain insight into the observed slow diffusion in yeast membranes, we explored lateral diffusion of two proteins of different origin. We compared lateral dynamics of the Candida drug resistance protein-1 (Cdr1p), and the human serotonin_1A receptor (5-HT_1AR) by fluorescence recovery after photobleaching (FRAP). Our results show that while Cdr1p-GFP displays slow diffusion, the diffusion of 5-HT_1AR-EYFP is significantly faster. Interestingly, upon ergosterol depletion, the mobility of Cdr1p-GFP did not exhibit appreciable change, while 5-HT_1AR-EYFP mobility showed an increase. On the other hand, upon actin cytoskeleton destabilization, the mobile fraction of 5-HT_1AR-EYFP showed considerable increase, while the mobility of Cdr1p-GFP was not altered. Our results represent the first report on the dynamics of the important drug resistance protein Cdr1p and provide novel insight on diffusion of membrane proteins in yeast membranes.     

Improved mass spectrometric identification of gel-separated hydrophobic membrane proteins after sodium dodecyl sulfate removal by ion-pair extraction

Separation and identification of hydrophobic membrane proteins is a major challenge in proteomics. Identification of such sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE)-separated proteins by peptide mass fingerprinting (PMF) via matrix-assisted laser desorption/ionization-time of flight (MALDI-TOF) is frequently hampered by the insufficient amount of peptides being generated and their low signal intensity. Using the seven helical transmembrane-spanning proton pump bacteriorhodopsin as model protein, we demonstrate here that SDS removal from hydrophobic proteins by ion-pair extraction prior to in-gel tryptic proteolysis leads to a tenfold higher sensitivity in mass spectrometric identification via PMF, with respect to initial protein load on SDS-PAGE. Furthermore, parallel sequencing of the generated peptides by electrospray ionization-mass spectrometry (ESI-MS) and tandem mass spectrometry (MS/MS) was possible without further sample cleanup. We also show identification of other membrane proteins by this protocol, as proof of general applicability.     

Differential extraction of hydrophobic proteins from chloroplast envelope membranes: a subcellular-specific proteomic approach to identify rare intrinsic membrane proteins

Identification of rare hydrophobic membrane proteins is a major biological problem that is limited by the specific biochemical approaches required to extract these proteins from membranes and purify them. This is especially true for membranes, such as plastid envelope membranes, that have a high lipid content, present a wide variety of specific functions and therefore contain a large number of unique, but minor, proteins. We have optimized a procedure, based on the differential solubilization of membrane proteins in chloroform/methanol mixtures, to extract and concentrate the most hydrophobic proteins from chloroplast envelope membrane preparations, while more hydrophilic proteins were excluded. In addition to previously characterized chloroplast envelope proteins, such as the phosphate/triose phosphate translocator, we have identified new proteins that were shown to contain putative transmembrane α-helices. Moreover, using different chloroform/methanol mixtures, we have obtained differential solubilization of envelope proteins as a function of their hydrophobicity. All the proteins identified were genuine chloroplast envelope proteins, most of them being localized within the inner membrane. Our procedure enables direct mapping (by classical SDS-PAGE) and identification of hydrophobic membrane proteins, whatever their isoelectric point was, that are minor components of specific subcellular compartments. Thus, it complements other techniques that give access to peripheral membrane proteins. If applied to various cell membranes, it is anticipated that it can expedite the identification of hydrophobic proteins involved in transport systems for ions or organic solutes, or it may act as signal receptors or to control metabolic processes and vesicle trafficking.     

Butanol recovery from aqueous solution into ionic liquids by liquid–liquid extraction

Biobutanol has currently attracted considerable attention as an alternative biofuel to the petroleum-derived fuel due to several advantages including high energy content, low water absorption and easy application to the existing gasoline infrastructure. However, its production has still faced many obstacles to overcome including lack of energy-efficient butanol separation process from fermentation broth. To solve this issue, the extraction behavior of butanol from aqueous media into a variety of imidazolium-based ionic liquids (ILs) was investigated by liquid–liquid extraction. To understand the effect of ILs properties, the solvent characteristics of ILs such as mutual solubility of feed solvent (water) and extraction solvent (IL), distribution coefficient of butanol between water and IL, selectivity, and extraction efficiency were correlated with hydrophobicity and polarity of ILs. The butanol distribution between ILs and water strongly depends on the hydrophobicity of anions of ILs followed by the hydrophobicity of cations of ILs. On the other hand, butanol extraction efficiency and selectivity depend on the polarity of ILs. Considering extraction efficiency and selectivity, [Tf₂N]-based ILs among the tested ILs showed to be the best extract solvent for the recovery of butanol from aqueous media. Among the studied ILs, [Omim][Tf₂N] showed the highest butanol distribution coefficient (1.939), selectivity (132) and extraction efficiency (74%) at 323.15 K, respectively.     

Amino acid composition of the membrane and aqueous domains of integral membrane proteins

To identify residues which might impart transport capability to the intramembranous regions of transport proteins, we surveyed available data for the 9991 amino acids contained in the aqueous and intramembranous regions of 24 integral membrane proteins: 10 transport (T) proteins and 14 nontransport (NT) proteins. Statistical comparison of percentage occurrence of each amino acid within T and NT samples provided a measure of "typical" composition of T and NT membrane-spanning regions, and showed that the residues partition into membrane and aqueous domains largely in accord with expectation from hydropathy indices. Comparison of aqueous and membrane domain composition between protein categories revealed a statistically similar distribution of residues in aqueous domains, but significant differences in membrane domains: seven residues (Asn, Asp, Gln, Glu, Phe, Pro, Tyr) were preferred in membrane regions of T proteins, and one (Val) was selectively excluded. Chemical and structural considerations suggested that three of these residues--Asn, Tyr, and Pro--are the most likely functional participants in transport processes.     

Peptide self-association in aqueous trifluoroethanol monitored by pulsed field gradient NMR diffusion measurements

Defining the self-association state of a molecule in solution can be an important step in NMR-based structure determination. This is particularly true of peptides, where there can be a relatively small number of long-range interactions and misinterpretation of an intermolecular NOE as an intramolecular contact can have a dramatic influence on the final calculated structure. In this paper, we have investigated the use of translational self-diffusion coefficient measurements to detect self-association in aqueous trifluoroethanol of three peptides which are analogues of the C-terminal region of human neuropeptide Y. Experimentally measured diffusion coefficients were extrapolated to D⁰, the limiting value as the peptide concentration approaches zero, and then converted to D_20,w, the diffusion coefficient after correction for temperature and the viscosity of the solvent. A decrease in D_20,w of about 16% was found for all three peptides in aqueous TFE (30% by volume) compared with water, which is in reasonable agreement with the expected decrease upon dimerisation, the presence of which was indicated by sedimentation equilibrium measurements. Apparent molecular masses of these peptides in both solutions were also calculated from their diffusion coefficients and similar results were obtained. Several potential internal standards, including acetone, acetonitrile, dimethylsulfoxide and dioxane, were assessed as monitors of solution viscosity over a range of trifluoroethanol concentrations. Compared with independent measurements of viscosity, acetonitrile was the most accurate standard among these four. The practical limitations of a quantitative assessment of peptide self-association from translational diffusion coefficients measured by PFGNMR, including the calculation of apparent molecular mass, are also discussed.     

Mixed solvent systems for recovery of ethanol from dilute aqueous solution by liquid-liquid extraction

Distribution coefficients and selectivities of a number of mixed solvent systems have been determined in order to assess their suitability in preferentially extracting ethanol from aqueous solution. The measured values of distribution coefficients and selectivities differ substantially from the values estimated by interpolating between the pure solvents.