双水相萃取结合液相色谱法分离蛋白质

建立了PEG/( NH4)2SO4双水相体系萃取富集,结合液相色谱分离分析多种蛋白质的方法.考察了无机盐种类和浓度、PEG分子量、pH值和温度等因素对双水相形成以及对细胞色素C、肌红蛋白、牛血清白蛋白、溶菌酶、胰蛋白酶分配行为的影响.结果表明,上述5种蛋白在室温、pH 3.5～9.0范围内,可在15％ PEG-4000/10％ (NH4)2SO4双水相体系中得到富集,且主要集中在下相.同样条件下,血清中的高丰度蛋白在上下相均有分配,下相分配量较大.通过双水相萃取分离蛋白质及对液相色谱一定时间段的色谱峰收集,可初步实现血清中高丰度蛋白质的分离去除.     

The Influence of Sodium Phosphate on Extraction Phenomena of Aqueous Two‐Phase Cationic/Anionic Surfactant Systems

The phase behavior of dodecyltrimethylammonium bromide (DTAB)/sodium dodecyl sulfonate (AS)/H₂O system in the presence and in the absence of sodium phosphate has been studied. Two kinds of aqueous two‐phase systems (ATPSs) were formed, one is ATPS‐A in which anionic surfactant is in excess, the other is ATPS‐C in which cationic surfactant is in excess. For the CTAB/AS/H₂O system, the addition of sodium phosphate changes the extraction phenomena of both ATPS‐A and ATPS‐C. For the DTAB/AS/H₂O system, the addition of sodium phosphate changes the extraction phenomena of ATPS‐C. For ATPS‐C, the addition of trivalent PO₄ ^3? results in a strong extraction effect of ATPS‐C to cationic water‐soluble dye methylene blue.     

Thermoresponsive hybrid hydrogel of oxidized nanocellulose using a polypeptide crosslinker

Thermoresponsive hybrid nanocellulose hydrogels were prepared from a mixture of oxidized nanocellulose and elastin-like polypeptide (ELP). Positively charged ELP was used as a polymeric crosslinker for conjugation with negatively charged nanocellulose. Hydrogel formation was triggered by a simple increase in temperature, and the hydrogel was reversibly returned to the liquid phase by decreasing temperature. Surface potential measurement confirmed the electrostatic properties of oxidized nanocellulose and ELP molecules. The surface morphology of hydrogels was observed by atomic force microscopy and field emission-scanning electron microscopy. Conformational changes in the ELP/nanocellulose hybrid were characterized by circular dichroism. The ELP/nanocellulose hybrid hydrogel was noncytotoxic and suitable for encapsulating cells, indicating its potential for biomedical applications.     

Solvent systems for countercurrent chromatography: an aqueous two phase liquid system based on a room temperature ionic liquid

A new aqueous two phase liquid system (ATPS) based on the ionic liquid 1-butyl-3-methyl imidazolium chloride (BMIM Cl), potassium dibasic phosphate (K(2)HPO(4)) and water was recently proposed in the literature. The full phase diagram of this ATPS was prepared and some tie lines were fully determined. It was compared to classical ATPSs based on polyethylene glycol with an average molecular mass of 1000 (PEG 1000) and 10,000 (PEG 10000) and K(2)HPO(4). Two countercurrent chromatography (CCC) columns, a hydrostatic Sanki and a J type hydrodynamic CCC columns were used to test the liquid phase retention of these ATPSs in all possible configurations. It was found that the BMIM Cl ATPS liquid phases were much easier to retain in the two CCC columns than the PEG 1000 ATPS phases. Using protein and alcohol solutes, it was established that the BMIM Cl ATPS has a polarity completely different from that of the PEG 1000 ATPS. For example, ovalbumin partitions equally between the two phases of the PEG 1000 ATPS (K(D)=1.4) when it is completely located in the BMIM Cl upper phase of the ionic liquid ATPS (K(D)=180). The discrimination factor of the ionic liquid system and its intrinsic hydrophobicity were respectively found three times higher and ten times lower than the respective values of the PEG 1000 ATPS.     

蛋白质在表面活性剂与高分子共组双水相体系中 的分配

高分子和正负离子表面活性剂混合物可形成一种新型双水相体系。研究蛋白质在溴化十二烷基三乙铵/十二烷基硫酸钠与聚氧乙烯(EO)-聚氧丙烯(PO)嵌段共聚物(EO~2~0PO~8~0)共组双水相体系中的分配。通过在高分子接上亲和配基,研究蛋白质在带有亲和配基高分子的双水相体系中的分配。将表面活性剂富集相稀释或加热高分子富集相,又可形成新的双水相体系,由此可进行蛋白质的多步分配。在蛋白质的分配完成之后,通过将表面活性剂富集相进一步稀释或将高分子富集相加热至高分子浊点以上可将表面活性剂和高分子与目标蛋白质分离。正负离子表面活性剂和高分子还可以循环使用。     

Microstructures of Aqueous Two‐Phase Systems Formed by Mixture of Polymer and Cationic‐Anionic Surfactants

Microstructure and phase behavior of decyltriethylammonium bromide (C₁₀NE)/sodium decylsulfonate (C₁₀SO₃)/poly(ethylene oxide) (PEO)/water quaternary systems were studied by freeze‐fracture transmission electron microscopy, small angle X‐ray diffraction, and dynamic light scattering methods. Aqueous two‐phase systems (ATPS) could be prepared by properly mixing the aqueous solution of PEO and equimolar mixed C₁₀NE and C₁₀SO₃. It was shown that the top phase of the ATPS was surfactant‐enriched and mainly composed of multi‐lamellar structure, while the bottom phase of the ATPS was polymer‐enriched in which some vesicles were observed.     

"Smart" biopolymer for a reversible stimuli-responsive platform in cell-based biochips

The rapid response of a smart material surface to external stimuli is critical for application to cell-based biochips. The sharp and controllable phase transition of elastin-like polypeptide (ELP) enabled reversible cell adhesion on the surface by changing the temperature or salt concentration in the system. First, ELP micropatterns were prepared on a glass surface modified into aldehyde. The lysine-containing ELP (ELP-K) was genetically synthesized from E. coli for conjugation with the aldehyde on the glass surface. The phase transition of ELP was monitored in PBS and cell culture media using UV-visible spectroscopy, and a significant difference in transition temperature (Tt) was observed between the two solution systems. The micropatterning of ELP on the glass surface was performed by microcontact printing a removable polymeric template on the aldehyde-glass followed by incubation in ELP-K aqueous solution. The ELP micropatterns were imaged with atomic force microscopy and showed a monolayer thickness of approximately 4 nm. Imaging from time-of-flight secondary ion mass spectroscopy confirmed that the ELP molecules were successfully immobilized on the highly resolved micropatterns. Cell attachment and detachment could be reversibly controlled on the ELP surfaces by external stimuli. The hydrophobic phase above Tt resulted in the adhesion of fibroblasts, while the detachment of cells was induced by lowering the incubation temperature below Tt. The smart properties of ELP were reliable and reproducible, demonstrating potential applications in cell-based microdevices.     

Aqueous two-phase systems for protein separation: a perspective

Aqueous two-phase systems (ATPS) that are formed by mixing a polymer (usually polyethylene glycol, PEG) and a salt (e.g. phosphate, sulphate or citrate) or two polymers and water can be effectively used for the separation and purification of proteins. The partitioning between both phases is dependent on the surface properties of the proteins and on the properties of the two phase system. The mechanism of partitioning is complex and not very easy to predict but, as this review paper shows, some very clear trends can be established. Hydrophobicity is the main determinant in the partitioning of proteins and can be measured in many different ways. The two methods that are more attractive, depending on the ATPS used (PEG/salt, PEG/polymer), are those that consider the 3-D structure and the hydrophobicity of AA on the surface and the one based on precipitation with ammonium sulphate (parameter 1/m*). The effect of charge has a relatively small effect on the partitioning of proteins in PEG/salt systems but is more important in PEG/dextran systems. Protein concentration has an important effect on the partitioning of proteins in ATPS. This depends on the higher levels of solubility of the protein in each of the phases and hence the partitioning observed at low protein concentrations can be very different to that observed at high concentrations. In virtually all cases the partition coefficient is constant at low protein concentration (true partitioning) and changes to a different constant value at a high overall protein concentration. Furthermore, true partitioning behavior, which is independent of the protein concentration, only occurs at relatively low protein concentration. As the concentration of a protein exceeds relatively low values, precipitation at the interface and in suspension can be observed. This protein precipitate is in equilibrium with the protein solubilized in each of the phases. Regarding the effect of protein molecular weight, no clear trend of the effect on partitioning has been found, apart from PEG/dextran systems where proteins with higher molecular weights partitioned more readily to the bottom phase. Bioaffinity has been shown in many cases to have an important effect on the partitioning of proteins. The practical application of ATPS has been demonstrated in many cases including a number of industrial applications with excellent levels of purity and yield. This separation and purification has also been successfully used for the separation of virus and virus-like particles.     

Effect of 1-hexanol on the phase behavior of SDS/CTAB/NaBr/H2O system

Abstract

The influence of 1-hexanol on the phase behavior of sodium dodecyl sulfate (SDS)/cetyltrimethyl ammonium bromide (CTAB)/NaBr/H₂O system has been systematically investigated in this paper. The results showed 1-hexanol effectively dissolved the precipitate formed by the CTAB and SDS surfactants, while liquid crystal (LC) and aqueous two phase system (ATPS) were formed in a wider range. When the molar ratio of 1-hexanol to surfactant is higher than 1, the precipitation in the system disappeared completely and was transformed into ATPS and LC, indicating that alcohol inserted at least evenly between every two surfactant molecules and hence effectively weakened the electrostatic interaction between the anionic and cationic surfactants and limited the formation of precipitation. Polarizing microscope (POM) with crossed polarizers was employed to investigate the textures of liquid crystals. It was shown that the existence of lamellar LC was confirmed by “Maltese crosses” textures. Additionally, we showed that the thermal stability of LC was promising. The ATPS and LC regions remained stable and changed slightly when the temperature was increased from 40 to 70?°C. The results indicated that ATPS and LC of the system were quiet resistant to temperature with the addition of 1-hexanol.     

Aqueous two-phase partitioning of milk proteins

Milk of transgenic pigs secreting recombinant human Protein C (rHPC) was used as a model system to determine the utility of aqueous two-phase extraction systems (ATPS) for the initial step in the purification of proteins from milk. The major challenges in purification of recombinant proteins from milk are removal of casein micelles (that foul processing equipment) and elimination of the host milk proteins from the final product. When milk was partitioned in ATPS composed of polyethylene glycol (PEG) and ammonium sulfate (AS), the phases were clarified and most of the caseins precipitated at the interphase. The partition coefficients of the major milk proteins and rHPC were dependent upon the molecular weight of the PEG used in the ATPS. Higher-partition coefficients of the major whey proteins, Β-lactoglobulin, and α-lactalbumin were observed in ATPS made up of lower molecular-weight PEG (1000 or 1450) as compared to systems using higher molecular-weight PEG. Lowering the pH of the ATPS from 7.5 to 6.0 resulted in increased precipitation of the caseins and decreased their concentration in both phases. rHPC had a partition coefficient of 0.04 in a system composed of AS and PEG 1450. The rHPC in pig milk was shown to be highly heterogenous by two-dimensional gel electrophoresis. The heterogeneity was owing to inefficient proteolytic processing of the single chain to the heterodimeric form and differences in glycosylation and other post-translational processing. Differential partitioning of the multiple forms of purified rHPC in the ATPS was not observed. rHPC after processing in ATPS was recovered in a clear phase free of most major milk proteins. ATPS are useful as the initial processing step in the purification of recombinant proteins from milk because clarification and enrichment is combined in a single step.     

Affinity partitioning of human antibodies in aqueous two-phase systems

The partitioning of human immunoglobulin (IgG) in a polymer-polymer and polymer-salt aqueous two-phase system (ATPS) in the presence of several functionalised polyethylene glycols (PEGs) was studied. As a first approach, the partition studies were performed with pure IgG using systems in which the target protein remained in the bottom phase when the non-functionalised systems were tested. The effect of increasing functionalised PEG concentration and the type of ligand were studied. Afterwards, selectivity studies were performed with the most successful ligands first by using systems containing pure proteins and an artificial mixture of proteins and, subsequently, with systems containing a Chinese hamster ovary (CHO) cells supernatant. The PEG/phosphate ATPS was not suitable for the affinity partitioning of IgG. In the PEG/dextran ATPS, the diglutaric acid functionalised PEGs (PEG-COOH) displayed great affinity to IgG, and all IgG could be recovered in the top phase when 20% (w/w) of PEG 150-COOH and 40% (w/w) PEG 3350-COOH were used. The selectivity of these functionalised PEGs was evaluated using an artificial mixture of proteins, and PEG 3350-COOH did not show affinity to IgG in the presence of typical serum proteins such as human serum albumin and myoglobin, while in systems with PEG 150-COOH, IgG could be recovered with a yield of 91%. The best purification of IgG from the CHO cells supernatant was then achieved in a PEG/dextran ATPS in the presence of PEG 150-COOH with a recovery yield of 93%, a purification factor of 1.9 and a selectivity to IgG of 11. When this functionalised PEG was added to the ATPS, a 60-fold increase in selectivity was observed when compared to the non-functionalised systems.     

Aqueous Two‐Phase System (ATPS) Containing Gemini (12‐3‐12,2Br−) and SDS I: Phase Diagram and Properties of ATPS

Two phases coexist in an aqueous system that contains the two surfactants cationic gemini 12‐3‐12,2Br? and anionic SDS. An aqueous two‐phase system (ATPS) is formed in a narrow region of the ternary phase diagram different from that of traditional aqueous cationic‐anionic surfactant systems. In that region, the molar ratio of gemini to SDS varies with the total concentration of surfactants. ATPS not only has higher stability but also has longer phase separation time for the new systems than that of the traditional system. Furthermore, the optical properties of ATPS are different at different total concentrations. All of these experimental observations can be attributed to the unique properties of gemini surfactant and the synergy between the cationic gemini surfactant and the anionic surfactant SDS.     

Photoswitchable Phase Separation and Oligonucleotide Trafficking in DNA Coacervate Microdroplets

Coacervate microdroplets produced by liquid–liquid phase separation have been used as synthetic protocells that mimic the dynamical organization of membrane‐free organelles in living systems. Achieving spatiotemporal control over droplet condensation and disassembly remains challenging. Herein, we describe the formation and photoswitchable behavior of light‐responsive coacervate droplets prepared from mixtures of double‐stranded DNA and an azobenzene cation. The droplets disassemble and reassemble under UV and blue light, respectively, due to azobenzene trans/cis photoisomerisation. Sequestration and release of captured oligonucleotides follow the dynamics of phase separation such that light‐activated transfer, mixing, hybridization, and trafficking of the oligonucleotides can be controlled in binary populations of the droplets. Our results open perspectives for the spatiotemporal control of DNA coacervates and provide a step towards the dynamic regulation of synthetic protocells.     

Performance of an aqueous two‐phase‐based countercurrent chromatographic system for horseradish peroxidase purification

Countercurrent chromatography (CCC) purification of horseradish peroxidase (HRP) from Armoracia rusticana root extracts was achieved by employing polymer‐phosphate aqueous two‐phase systems (ATPS). By using preparative columns at 1000 rpm, a 25–30% retention of the top phase of an ATPS composed of 10% w/w PEG 1540 and 14.8% w/w phosphate – with added 2 mol/kg sodium chloride – was obtained. The retention level was stable during the standard separation running time (4 h). Horseradish root extract samples were injected into the system (10–25 mL; 200–250 U/mL peroxidase; 2.0–4.0 mg/mL total protein). Retention of HRP in the CCC “column” during the chromatographic run was attained in the selected ATPS, where the partition coefficient K for the enzyme was ≥ 8. Replacement of the mobile phase with a fresh one but in the absence of added salt brought about product elution. Recovery of HRP in this fraction accounts for ≥ 45% of the total activity loaded, with a purification factor of 6. Enzyme activity was also found in the pass‐through fraction and in the remaining liquid (stationary) phase, a fact that should be ascribed to the existence of multiple peroxidase isoforms. SDS‐PAGE of the active fraction showed a protein band at 44 kDa, compatible with the presence of HRP. Thus, the optimised CCC system allowed the separation of HRP directly from a complex biological material. These results open up the possibility of achieving protein separation with CCC/ATPS and of scaling‐up processes in industrial separators.     

Extraction Separation of BSA in Aqueous Two-Phase Systems of Anionic and Cationic Surfactant Mixtures

Aqueous two-phase systems (ATPS) are formed in mixtures of sodium dodecylsulfate (SDS), cetyltrimethyl ammonium bromide (CTAB), and sodium bromide (NaBr). Two different kinds of ATPS appear when CTAB and SDS surfactants are in excess, respectively. Such ATPS may provide a new, useful partitioning system for separation of bovine serum albumin (BSA). The partitioning of BSA was studied in these systems. The results show that the partitioning is affected by the electrical property, the hydrophobicity and the structure of liquid crystal (LC), which exists in the top phase of the ATPS. BSA is extracted into the top phase with higher distribution coefficient when LC and BSA are oppositely charged. The hydrophobicity of LC, which can be improved by increasing the length of alkyl group, enhances the distribution coefficient. The hydrophobicity of lamellar LC and hexagonal LC is stronger than that of cubic LC, which causes extractive capability of the former is higher than the latter.     

The influence of temperature on the phase behavior of ionic liquid aqueous two-phase systems

In this article, the equilibrium behavior of solutions of 1-ethyl-3-methylimidazolium dimethyl phosphate ([Emim]DMP) and ethyl acetate or acetone in aqueous two-phase system (ATPS) was discussed to understand the liquid–liquid equilibrium (LLE) behavior of these organic solvents. Thus, we determined phase diagrams and LLE data at 303.15, 308.15, 313.15, and 323.15 K for the investigated biphasic systems. Four empirical equations were used to study the tie lines. The results showed that for the [Emim]DMP?+?acetone?+?water biphasic systems within the investigated temperature range, temperature influences the phase behavior, but for the [Emim]DMP?+?ethyl acetate?+?water biphasic systems within the investigated temperature range, there are no notable changes on the phase behavior with rising temperature. The results may have important applications for the separation of antibiotics and for the recovery of ionic liquids (ILs).     

Spatial Positioning and Chemical Coupling in Coacervate‐in‐Proteinosome Protocells

The integration of molecularly crowded microenvironments into membrane‐enclosed protocell models represents a step towards more realistic representations of cellular structure and organization. Herein, the membrane diffusion‐mediated nucleation of either negatively or positively charged coacervate microdroplets within the aqueous lumen of individual proteinosomes is used to prepare nested hybrid protocells with spatially organized and chemically coupled enzyme activities. The location and reconfiguration of the entrapped droplets are regulated by tuning the electrostatic interactions between the encapsulated coacervate and surrounding negatively charged proteinosome membrane. As a consequence, alternative modes of a cascade reaction involving membrane‐ and coacervate‐segregated enzymes can be implemented within the coacervate‐in‐proteinosome protocells.     

Evaluation of a PEG/hydroxypropyl starch aqueous two‐phase system for the separation of monoclonal antibodies from cell culture supernatant

In this study, an aqueous two‐phase system (ATPS) with PEG and hydroxypropyl starch (HPS) was used to separate monoclonal antibody (mAb) from Chinese hamster ovary cell culture supernatant. The phase diagram of the PEG/HPS ATPS was determined, and the effects of NaCl addition were investigated. The results showed that NaCl addition could lead to a shift of the binodal curve and that phase separation would occur at higher PEG and HPS concentrations. The effects of NaCl addition, pH, and the load of cell supernatant on the partitioning of mAb in a PEG/HPS ATPS were investigated. It was found that with 6% cell supernatant and 15% NaCl addition at pH 6.0, the yield of mAb in the upper phase was 96.7% with a purity of 96.0%. The back‐extraction of mAb with a PEG/phosphate ATPS were also studied, and the results showed that after the two‐step extraction with ATPSs the purity of mAb could reach 97.6 ± 0.5% with a yield of 86.8 ± 1.0%, which was comparable to the purification with Protein A chromatography. These results indicate that the two‐step extraction with PEG/HPS and PEG/phosphate ATPSs might be a promising alternative for the separation of mAb from cell culture supernatant.     

Rheology and microstructure studies of SDS/CTAB/H2O system

Phase behavior of mixed sodium dodecyl sulfate (SDS) and cetyl trimethyl ammonium bromide (CTAB) aqueous solution was studied. The rheological properties and microstructure were investigated using a rheostat and freeze-fracture technique and are shown to be closely related to the phase behavior. Experimental investigations reveal two symmetrical aqueous two-phase systems (ATPS) in the ternary phase diagram of SDS/CTAB/H₂O system. In the surfactant rich phase of ATPS or in the adjacent stoichiometric state of ATPS, the system has high viscosity because of its long range ordered structure. Lamellar phase was found in the high viscosity samples in which the cationic and anionic surfactant are in 1: 3 or 3: 1 stoichiometry. In addition, the viscosity has a tendency to increase when salt was added to the solution. The viscosity increase is due to the salt can screen the repulsion between different charged headgroups and thus reduces the effective size of surfactants and facilitates the spherical or rod likes micelles to be transformed to worm-like micelles which can form hexagonal or liquid crystal phases. Large-size salt ions like sodium sulfate (especially organic salt ions) have more significant effect on the surfactant solution viscosity. The text was submitted by the authors in English.     

一种用于鸡蛋中氯霉素残留测定的吡啶类离子液体双水相体系

建立了由亲水性离子液体N-乙基吡啶溴化盐([Epy]Br)和K2HPO4形成的双水相体系对鸡蛋中痕量氯霉素(CAP)检测的高效液相色谱方法.优化了离子液体[Epy]Br和K2HPO4成相的条件,研究了萃取CAP的最佳体系.采用混合溶液(pH=7磷酸盐溶液,NaCl溶液,甲醇)提取样品,离子液体双水相体系富集,V(甲醇):V(水)=30:70为流动相,流速1.0 mL/min,UV检测器,检测波长278 nm,柱温25 ℃,进样量20 μL.对鸡蛋中氯霉素残留进行了测定,在0.05～15 mg/L范围内,CAP的响应峰面积与其相应浓度呈良好相关性(r=0.9994).不同浓度的回收率为90.3%～101.7%,相对标准偏差为1.7%～2.6%.本该法检测鸡蛋中氯霉素残留具有干扰小、速度快、灵敏度高等优点.最低检出限为1 μg/kg.