Genetically engineered peptide fusions for improved protein partitioning in aqueous two-phase systems. Effect of fusion localization on endoglucanase I of Trichoderma reesei

Genetic engineering has been used for fusion of the peptide tag, Trp-Pro-Trp-Pro, on a protein to study the effect on partitioning in aqueous two-phase systems. As target protein for the fusions the cellulase, endoglucanase I (endo-1,4-beta-Dglucan-4-glucanohydrolase, EC 3.2.1.4, EGI, Cel7B) of Trichoderma reesei was used. For the first time a glycosylated two-domain enzyme has been utilized for addition of peptide tags to change partitioning in aqueous two-phase systems. The aim was to find an optimal fusion localization for EGI. The peptide was (1) attached to the C-terminus end of the cellulose binding domain (CBD), (2) inserted in the glycosylated linker region, (3) added after a truncated form of EGI lacking the CBD and a small part of the linker. The different constructs were expressed in the filamentous fungus T. reesei under the gpdA promoter from Aspergillus nidulans. The expression levels were between 60 and 100 mg/l. The partitioning behavior of the fusion proteins was studied in an aqueous two-phase model system composed of the thermoseparating ethylene oxide (EO)-propylene oxide (PO) random copolymer EO-PO (50:50) (EO50PO50) and dextran. The Trp-Pro-Trp-Pro tag was found to direct the fusion protein to the top EO50PO50 phase. The partition coefficient of a fusion protein can be predicted with an empirical correlation based on independent contributions from partitioning of unmodified protein and peptide tag in this model system. The fusion position at the end of the CBD, with the spacer Pro-Gly, was shown to be optimal with respect to partitioning and tag efficiency factor (TEF) was 0.87, where a fully exposed tag would have a TEF of 1.0. Hence, this position can further be utilized for fusion with longer tags. For the other constructs the TEF was only 0.43 and 0.10, for the tag fused to the truncated EGI and in the linker region of the full length EGI, respectively.     

Direct purification of pectinase from mango (Mangifera Indica Cv. Chokanan) peel using a PEG/salt-based Aqueous Two Phase System

An Aqueous Two-Phase System (ATPS) was employed for the first time for the separation and purification of pectinase from mango (Mangifera Indica Cv. Chokanan) peel. The effects of different parameters such as molecular weight of the polymer (polyethylene glycol, 2,000-10,000), potassium phosphate composition (12-20%, w/w), system pH (6-9), and addition of different concentrations of neutral salts (0-8%, w/w) on partition behavior of pectinase were investigated. The partition coefficient of the enzyme was decreased by increasing the PEG molecular weight. Additionally, the phase composition showed a significant effect on purification factor and yield of the enzyme. Optimum conditions for purification of pectinase from mango peel were achieved in a 14% PEG 4000-14% potassium phosphate system using 3% (w/w) NaCl addition at pH 7.0. Based on this system, the purification factor of pectinase was increased to 13.2 with a high yield of (97.6%). Thus, this study proves that ATPS can be an inexpensive and effective method for partitioning of pectinase from mango peel.     

Two-step recovery process for tryptophan tagged cutinase: interfacing aqueous two-phase extraction and hydrophobic interaction chromatography

In this work, the interfacing of a poly(ethylene glycol) (PEG)-phosphate aqueous two-phase system with hydrophobic interaction chromatography (HIC) for primary recovery of an intracellular protein was evaluated. As a model protein, a recombinant cutinase furnished with a tryptophan-proline (WP) peptide tag was used and produced intracellularly in Escherichia coli (E. coli). E. coli cell homogenate was partitioned in a two-phase system and the top phase yield, concentration and purity of the tagged ZZ-cutinase-(WP)4 was evaluated as function of polymer sizes, system pH and phase volume ratio. The partition behaviour of cell debris, total protein and endotoxin was also monitored. In the HIC part, the chromatographic yield and purity was investigated with respect to ligand hydrophobicity, dilution of loaded top phase and elution conditions. Based on the results, a recovery process was demonstrated where a PEG 1500-K-Na phosphate salt aqueous two-phase system was interfaced with a HIC column. The interfacing was facilitated by the Trp-tagged peptide. The tagged ZZ-cutinase-(WP)4 was obtained in a PEG-free phase and purified to >95% purity according to silver stained sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) gels with a total yield of 83% during the two-step recovery process.     

Partitioning of Porcine Pancreatic Lipase in a Two-Phase Systems of Polyethylene Glycol/Potassium Phosphate Aqueous

The hydrolysis of triglycerides at the oil–water interface, synthesis of esters and transesterification in microaqueous conditions are catalysed by lipase. For its application, a proper purification method was necessary. This study examined the application of an aqueous two-phase system to partition porcine pancreatic lipase. The influence of molecular weight and concentration of polyethylene glycol (PEG), tie line length (TLL), potassium phosphate concentration, sodium chloride (NaCl) addition and temperature in the partition was studied. The enzyme was more efficiently purified in PEG 8,000 at 14.5 °C (PF?=?3.89-fold), presenting more recoveries at the top phase with shorter TLL and lower concentrations of PEG and potassium phosphate. Moreover, the increase of these variables repressed the purification and the further addition of NaCl did not promote the purification of the enzyme. These results demonstrated the efficiency of the aqueous two-phase system on lipase purification.     

Horseradish peroxidase extraction and purification by aqueous two-phase partition

The effect of poly(ethyleneglycol) (PEG) molecular weight, system pH, and sodium chloride concentration on the partitioning behavior of horseradish peroxidase fromArmomcia rusticana root extract was investigated in poly(ethyleneglycol)/sodium phosphate systems. PEG molecular weight strongly affects the enzyme partition coefficient, whereas pH variation from 5.5 to 8.0 has little effect. The addition of sodium chloride (8% w/w) to a PEG 1540/phosphate system, pH 7.0, raises the peroxidase partition coefficient 13.5-fold without important changes in that of total horseradish root proteins. Moreover, these conditions allow direct homogenization of theA. rusticana roots with the selected aqueous two-phase system with the clear top phase containing over 90% of the enzyme and the purification factor being 4.8.     

Genetically engineered peptide fusions for improved protein partitioning in aqueous two-phase systems: Effect of fusion localization on endoglucanase I of Trichoderma reesei

Genetic engineering has been used for fusion of the peptide tag, Trp–Pro–Trp–Pro, on a protein to study the effect on partitioning in aqueous two-phase systems. As target protein for the fusions the cellulase, endoglucanase I (endo-1,4-β- -glucan-4-glucanohydrolase, EC 3.2.1.4, EGI, Cel7B) of Trichoderma reesei was used. For the first time a glycosylated two-domain enzyme has been utilized for addition of peptide tags to change partitioning in aqueous two-phase systems. The aim was to find an optimal fusion localization for EGI. The peptide was (1) attached to the C-terminus end of the cellulose binding domain (CBD), (2) inserted in the glycosylated linker region, (3) added after a truncated form of EGI lacking the CBD and a small part of the linker. The different constructs were expressed in the filamentous fungus T. reesei under the gpdA promoter from Aspergillus nidulans. The expression levels were between 60 and 100 mg/l. The partitioning behavior of the fusion proteins was studied in an aqueous two-phase model system composed of the thermoseparating ethylene oxide (EO)–propylene oxide (PO) random copolymer EO–PO (50:50) (EO₅₀PO₅₀) and dextran. The Trp–Pro–Trp–Pro tag was found to direct the fusion protein to the top EO₅₀PO₅₀ phase. The partition coefficient of a fusion protein can be predicted with an empirical correlation based on independent contributions from partitioning of unmodified protein and peptide tag in this model system. The fusion position at the end of the CBD, with the spacer Pro–Gly, was shown to be optimal with respect to partitioning and tag efficiency factor (TEF) was 0.87, where a fully exposed tag would have a TEF of 1.0. Hence, this position can further be utilized for fusion with longer tags. For the other constructs the TEF was only 0.43 and 0.10, for the tag fused to the truncated EGI and in the linker region of the full length EGI, respectively.     

Xylanase recovery effect of extraction conditions on the aqueous two-phase system using experimental design

The partitioning of xylanase produced byPenicillium janthinellum in aqueous two-phase systems (ATPS) using poly(ethylene glycol) (PEG) and phosphate (K₂HPO₄/KH₂PO₄) was studied employing a statistical experimental design. The aim was to identify the key factors governing xylanase partitioning. The interactions of five factors (PEG concentration molecular weight, concentration of buffer K₂HPO₄/KH₂PO₄, pH, and NaCl concentration) and their main effects on the partition coefficient (K) were evaluated by means of a 2⁵ full-factorial experimental design with four center points. The %PEG, %NaCl, and pH were the most important factors affecting the response variable (K). Response surface methodology (RSM) was adopted and an empirical second-order polynomial model was constructed on the basis of the results. The optimum partition conditions were pH 7.0, PEG = 8.83% and NaCl = 6.02%. Adequacy of the model for predicting optimum response value was tested under these conditions. The experimental xylanase partition coefficient (K) was 2.21, whereas its value predicted by the model was 2.33. These results indicate that the predicted model was adequate for the process. PEG molecular weight and phosphate concentration did not affect the xylanase partition coefficient.     

One-step purification of alpha-amylase from the cultivation supernatant of recombinant bacillus subtilis by high-speed counter-current chromatography with aqueous polymer two-phase systems

Purification of alpha-amylase from the cultivation supernatant of recombinant Bacillus subtilis by high-speed counter-current chromatography (HSCCC) in polyethylene glycol (PEG) 4000-inorganic salt aqueous polymer two-phase systems was studied. The effects of sodium chloride concentration on the partition coefficients of alpha-amylase and total protein were respectively tested in PEG4000-phosphate and PEG4000-citrate aqueous polymer two-phase systems to find the proper range of sodium chloride concentration for the HSCCC purification of alpha-amylase. Alpha-amylase was purified from the cultivation supernatant by HSCCC in PEG4000-phosphate system containing 2% (w/w) sodium chloride, yet with considerable loss of activity. PEG4000-citrate aqueous polymer two-phase system containing 2% (w/w) sodium chloride and supplemented with 0.56% (w/w) CaCl2 as protective agent was then successfully applied to purify alpha-amylase from cultivation supernatant by HSCCC to homogeneity and significantly increased the recovery of alpha-amylase activity from around 30 to 73.1%.     

Influence of poly(ethylene glycol) on the partition of a charged spin probe

The influence of poly(ethylene glycol) on the partition of a charged long chain spin probe between membranes and an external phase is studied. The partition coefficient is derived from the ESR spectra. Membranes of different properties are used (egg lecithin liposomes, erythrocytes) to differentiate between the influence of the external phase and the specific properties of the membrane.The partition coefficient is decreased in an exponential manner on increasing the PEG concentration, which indicates a lowering of the thermodynamic stability of the membranes. The determination of the change in the difference of the chemical potential is dependent on the PEG concentration.The membrane destabilization induced by PEG is caused in an indirect manner by a change of the chemical potential difference as result of the changed water structure and the osmotic pressure, surface tension and hydration of the membrane. This destabilization could be connected with the high fusogenic activity of PEG.     

聚丙烯酰胺-聚乙二醇-水体系相图及丙烯酰胺单体在两相中的分配

聚丙烯酰胺(PAAm)和聚乙二醇(PEG)两种水溶液混合时能形成双水相体系,其中上层为PEG富集相,下层为PAAm和PEG的混合相.用凝胶渗透色谱(GPC)法和浊度滴定法研究了PAAm-PEG-H₂O双水相体系的相图,结果表明,随着PEG分子量的升高,体系的分相浓度下降.在PAAm-PEG20000-H₂O体系中,随着体系温度升高,分相浓度先下降后升高,55℃时分相浓度最低.丙烯酰胺(AAm)单体能在两相中发生相分配,分配系数随着PAAm浓度和平衡温度的增加而增大,随着PEG浓度的增加而下降.     

Selective separation and enrichment of proteins in aqueous two-phase extraction system

A simple aqueous two-phase extraction system(ATPS) of PEG/phosphate was proposed for selective separation and enrichment of proteins.The combination of ATPE with HPLC was applied to identify the partition of proteins in two phases.Five proteins (bovine serum albumin,Cytochrome C,lysozyme,myoglobin,and trypsin) were used as model proteins to study the effect of phosphate concentration and pH on proteins partition.The PEG/phosphate system was firstly applied to real human saliva and plasma samples,some pro...     

Partitioning of l-methionine in aqueous two-phase systems containing poly(propylene glycol) and sodium phosphate salts

The partitioning behavior of l-methionine has been studied in aqueous two-phase systems of (poly(propylene glycol) + sodium phosphate salts + H₂O) at different temperatures. The salts used were sodium di-hydrogen phosphate (NaH₂PO₄), di-sodium hydrogen phosphate (Na₂HPO₄) and tri-sodium phosphate (Na₃PO₄). The effects of tie line length, salt type, and temperature on the partition coefficient of this amino acid have been studied. In addition, thermodynamic parameters (ΔH°, ΔS° and ΔG°) as a function of temperature were calculated. The results showed that increasing tie line length led to decreasing of the partition coefficient. We also showed that the partition coefficients of the amino acid in the systems containing Na₃PO₄ are greater than the other two salts. Moreover, it is verified that increasing temperature led to decreasing the partition coefficient. The experimental partition coefficient data are correlated using a modified virial-type model.     

Affinity partitioning of plasmid DNA with a zinc finger protein

The affinity isolation of pre-purified plasmid DNA (pDNA) from model buffer solutions using native and poly(ethylene glycol) (PEG) derivatized zinc finger-GST (Glutathione-S-Transferase) fusion protein was examined in PEG-dextran (DEX) aqueous two-phase systems (ATPSs). In the absence of pDNA, partitioning of unbound PEGylated fusion protein into the PEG-rich phase was confirmed with 97.5% of the PEGylated fusion protein being detected in the PEG phase of a PEG 600-DEX 40 ATPS. This represents a 1322-fold increase in the protein partition coefficient in comparison to the non-PEGylated protein (Kc = 0.013). In the presence of pDNA containing a specific oligonucleotide recognition sequence, the zinc finger moiety of the PEGylated fusion protein bound to the plasmid and steered the complex to the PEG-rich phase. An increase in the proportion of pDNA that partitioned to the PEG-rich phase was observed as the concentration of PEGylated fusion protein was increased. Partitioning of the bound complex occurred to such an extent that no DNA was detected by the picogreen assay in the dextran phase. It was also possible to partition pDNA using a non-PEGylated (native) zinc finger-GST fusion protein in a PEG 1000-DEX 500 ATPS. In this case the native ligand accumulated mainly in the PEG phase. These results indicate good prospects for the design of new plasmid DNA purification methods using fusion proteins as affinity ligands.     

温控聚乙二醇两相体系中纳米钯催化肉桂醛选择性加氢反应

将具有“高温混溶、室温分相”功能的聚乙二醇4000(PEG₄₀₀₀)与甲苯-正庚烷组成的两相体系用于纳米钯催化的肉桂醛选择性加氢反应中.在优化的反应条件下,肉桂醛转化率和氢化肉桂醛选择性分别为99%和98%.钯纳米催化剂经简单分相即可与产物分离,且循环使用8次,其活性和选择性基本保持不变.     

'Heat-treatment aqueous two phase system' for purification of serine protease from Kesinai (Streblus asper) leaves

A 'Heat treatment aqueous two phase system' was employed for the first time to purify serine protease from kesinai (Streblus asper) leaves. In this study, introduction of heat treatment procedure in serine protease purification was investigated. In addition, the effects of different molecular weights of polyethylene glycol (PEG 4000, 6000 and 8000) at concentrations of 8, 16 and 21% (w/w) as well as salts (Na-citrate, MgSO? and K?HPO?) at concentrations of 12, 15, 18% (w/w) on serine protease partition behavior were studied. Optimum conditions for serine protease purification were achieved in the PEG-rich phase with composition of 16% PEG6000-15% MgSO?. Also, thermal treatment of kesinai leaves at 55 °C for 15 min resulted in higher purity and recovery yield compared to the non-heat treatment sample. Furthermore, this study investigated the effects of various concentrations of NaCl addition (2, 4, 6 and 8% w/w) and different pH (4, 7 and 9) on the optimization of the system to obtain high yields of the enzyme. The recovery of serine protease was significantly enhanced in the presence of 4% (w/w) of NaCl at pH 7.0. Based on this system, the purification factor was increased 14.4 fold and achieved a high yield of 96.7%.     

Application of aqueous mixtures of polypropylene glycol or polyethylene glycol with salts in proteomic analysis

Partitioning of Bovine serum albumin (BSA), β-lactoglobulin (β-LG) and zein as model proteins in aqueous two-phase systems (S) containing polypropylene glycol (PPG425) or polyethylene glycol (PEG 6000) and salts (MgSO₄, (NH₄)₂SO₄, Na₂SO₄) is presented in this paper. The effects of different factors such as tie-line length, salt type and polymer type on the partition coefficient and recovery percent of proteins were analysed. The model proteins were separated by these systems (S) and directly used for gel electrophoresis without separating the target proteins from phase-forming reagents. The results revealed that the S, studied in this work could be used as a novel prefractionation method in proteomic analysis and could separate proteomic proteins in multigroup by one step extraction.     

Prediction of the partitioning behaviour of proteins in aqueous two-phase systems using only their amino acid composition

The prediction of the partition behaviour of proteins in aqueous two-phase systems (ATPS) using mathematical models based on their amino acid composition was investigated. The predictive models are based on the average surface hydrophobicity (ASH). The ASH was estimated by means of models that use the three-dimensional structure of proteins and by models that use only the amino acid composition of proteins. These models were evaluated for a set of 11 proteins with known experimental partition coefficient in four-phase systems: polyethylene glycol (PEG) 4000/phosphate, sulfate, citrate and dextran and considering three levels of NaCl concentration (0.0% w/w, 0.6% w/w and 8.8% w/w). The results indicate that such prediction is feasible even though the quality of the prediction depends strongly on the ATPS and its operational conditions such as the NaCl concentration. The ATPS 0 model which use the three-dimensional structure obtains similar results to those given by previous models based on variables measured in the laboratory. In addition it maintains the main characteristics of the hydrophobic resolution and intrinsic hydrophobicity reported before. Three mathematical models, ATPS I-III, based only on the amino acid composition were evaluated. The best results were obtained by the ATPS I model which assumes that all of the amino acids are completely exposed. The performance of the ATPS I model follows the behaviour reported previously, i.e. its correlation coefficients improve as the NaCl concentration increases in the system and, therefore, the effect of the protein hydrophobicity prevails over other effects such as charge or size. Its best predictive performance was obtained for the PEG/dextran system at high NaCl concentration. An increase in the predictive capacity of at least 54.4% with respect to the models which use the three-dimensional structure of the protein was obtained for that system. In addition, the ATPS I model exhibits high correlation coefficients in that system being higher than 0.88 on average. The ATPS I model exhibited correlation coefficients higher than 0.67 for the rest of the ATPS at high NaCl concentration. Finally, we tested our best model, the ATPS I model, on the prediction of the partition coefficient of the protein invertase. We found that the predictive capacities of the ATPS I model are better in PEG/dextran systems, where the relative error of the prediction with respect to the experimental value is 15.6%.     

Candida rugosa lipase Lip1–polyethyleneglycol interaction and the relation with its partition in aqueous two-phase systems

The interaction between a lipase from Candida rugosa (Lip1) and polyethyleneglycols of different molecular masses was studied using fluorescence and circular dichroism approaches in order to be applied to the analysis of the enzyme partition mechanism in aqueous two-phase systems of polyethyleneglycol–potassium phosphate. The decrease of the partition coefficients with the polyethyleneglycol molecular mass showed that the enzyme partition is driven by the excluded volume effect and not by the enzyme–polymer interaction. The polymer did not affect the secondary and tertiary structure of the enzyme nor its biological activity. The lipase from Candida rugosa lyophilizate was partitioned in favour of the polyethyleneglycol rich phase; PEG 2000 being the system which showed the better enzyme recovery (78.26%) with a purification factor of 2.3. This method could be applied as a first step to isolate the enzyme from a culture medium with good recovery and without modifying the enzymatic capacity and the molecular structure.     

Quantitative analysis of biodegradable amphiphilic poly(L-lactide)-block-poly(ethyleneglycol)-blockpoly(L-lactide) by using TG, FTIR and NMR

The thermogravimetric analysis (TG) of two series of tri-block copolymers based on poly(L,L-lactide) (PLLA) and poly(ethyleneglycol) (PEG) segments, having molar mass of 4000 or 600 g mol^–1, respectively, is reported. The prepared block copolymers presented wide range of molecular masses (800 to 47500 g mol^–1) and compositions (16 to 80 mass% PEG). The thermal stability increased with the PLLA and/or PEG segment size and the tri-block copolymers prepared from PEG 4000 started to decompose at higher temperatures compared to those copolymers from PEG 600. The copolymers compositions were determined by thermogravimetric analysis and the results were compared to other traditional quantitative spectroscopic methods, hydrogen nuclear magnetic resonance spectrometry (¹HNMR) and Fourier transform infrared spectrometry (FTIR). The PEG 4000 copolymer compositions calculated by TG and by ¹HNMR, presented differences of 1%, demonstrating feasibility of using thermogravimetric analysis for quantitative purposes.     

SYNTHESIS OF NOVEL BLOCK COPOLYMERS OF POLY(3-HYDROXYBUTYRIC ACID)WITH POLY(ETHYLENE GLYCOL)THROUGH ANIONIC POLYMERISATION

A novel kind of copolymer with ABA-type block structure was synthesized by anionic ring-openingpolymerization of β-butyrolactone (β-BL) in the presence of a PEG-based dicarboxylates as macroinitiators which wereprepared by the esterification of aliphatic cyclic anhydride and poly(ethylene glycol) (PEG) oligomers (M_n=2000, 4000 and6000) and conversion of potassium dicarboxylates. The resultant copolymers as well as the intermediates were characterizedby IR,~1H-NMR and GPC.