Extraction Of Bovine Serum Albumin Research Articles

Interactions between deep eutectic solvents and bovine serum albumin studied by spectroscopic and electrochemical methods

Deep eutectic solvent (DES) has become a potential protein extractant as its good biocompatibility, environmental friendliness and accessibility. In this study, two types of DESs, ferric chloride hexahydrate: oxalic acid (FeCl3·6H2O: H2C2O4) and choline chloride: zinc chloride (ChCl: ZnCl2), were prepared. The interactions between these two types of DESs and bovine serum albumin (BSA) were investigated by utilizing spectroscopic and electrochemical methods. It was found that both FeCl3·6H2O: H2C2O4 and ChCl: ZnCl2 are able to form complexes with BSA by a relatively high affinity. During the process, no significant change in BSA conformation was observed. The binding ratios of BSA in the FeCl3·6H2O: H2C2O4 and ChCl: ZnCl2 systems are up to 98.59 % and 98.14 %, respectively, indicating that the two DESs are both promising extractants for BSA. The interactions between the DESs and BSA are mainly hydrogen bond and van der Waals force, which are strong enough to extract BSA but not too strong for regeneration, making the DESs more applicable in the industry. Moreover, it was found that the binding ratio of BSA in the FeCl3·6H2O: H2C2O4-BSA system remains above 91.7 % in the temperature range of 298–313 K, illustrating the potential of FeCl3·6H2O: H2C2O4 to be used in BSA extraction under a wider range of temperature. In contrast, ChCl: ZnCl2 will be better used under lower temperatures.

Extraction of Bovine Serum Albumin by Aqueous Two-Phase System Using PEG4000/Sodium Citrate and PEG8000/Sodium Phosphate

Aqueous Two Phase System (ATPS) or liquid-liquid extraction is used in biotechnology to recover valuable compounds from raw sources. In Aqueous Two-Phase Systems, many factors influence the Partition coefficient, K, (which is the ratio of protein concentration in the top phase to that in the bottom phase) and the Recovery percentage (Rec%). In this research, two systems of ATPS were used: first, polyethylene glycol (PEG) 4000/Sodium citrate (SC), and the second, PEG8000/ Sodium phosphate (SPH), for the extraction of Bovine Serum Albumin (BSA). The behavior of Rec% and K of pure (BSA) in ATPS has been investigated throughout the study by the effects of five parameters: temperature, concentration of polyethylene glycol (PEG4000 and PEG8000), the concentration of Sodium citrate or Sodium phosphate, pH, and the addition of sodium chloride as a supporting agent. The recovery percentage of BSA and its partition coefficient are significantly influenced by these factors to various degrees. The most influential variable in this study is PEG concentration for both systems. In addition to the PEG concentration, the stabilizing impact of NaCl is a crucial factor. The interaction between biomolecules and PEG gets more hydrophobic as the PEG concentration is raised. In the first system (PEG4000/SC), the maximum recovery percentage and partition coefficient were 98.99% and 97.69, respectively, at 31°C, PEG4000 concentration 1.5g/10 ml, Sodium citrate concentration 2.7 g/10 ml, pH 10, and 0.5 M NaCl concentration. While in the second system (PEG8000/SPH), the maximum recovery percentage and partition coefficient was 98.93% and 92.12, respectively, at 31oC, PEG8000 concentration 1.5 g/10 ml, Sodium phosphate concentration 2.4 g/10 ml, pH 10, and concentration of NaCl 0.5 M.

Combination of deep eutectic solvent and organic–inorganic hybrid monomer to prepare monolith for improvement of hydrophilic protein extraction

Two kinds of monomers based on deep eutectic solvents (DESs) and 3-aminopropyl triethoxysilane-methacrylic acid (APTES-MAA) have been synthesized and used to prepare hybrid monoliths for hydrophilic protein extraction. A typical polymerization system consists of APTES-MAA (hybrid monomer), choline chloride/methacrylic acid (MAA) [DES monomer]), and ethylene glycol dimethacrylate (EDMA) in the presence of binary porogens of PEG 20,000 and methanol. The combined effect of the DES monomer and hybrid monomer in the monolithic column material was demonstrated by the recovery of bovine serum albumin (BSA). Compared with the DES/APTES-MAA-free monolith, the poly(APTES-MAA-co-DES-co-EDMA) monolith displayed a higher recovery of 95.5% (relative standard deviation < 5.0%). Monolith-characterization studies including field emission scanning electron microscopy, nitrogen adsorption, and thermal gravimetric analysis showed that the simultaneous use of DES and hybrid monomer to prepare monolith formed a unique structure. The study on the affinity of the monolithic column to protein was carried out by extracting BSA, ovalbumin (OVA), cytochrome c (Cyt c), avidin, and horseradish peroxidase (HRP). Experimental results demonstrated that the proposed poly(APTES-MAA-co-DES-co-EDMA) monolith performed significantly better in the extraction of hydrophilic proteins (BSA, Cyt c, and avidin) than the hydrophobic protein (HRP) and amphiphilic protein (OVA). The separation performance of poly(APTES-MAA-co-DES-co-EDMA) monolith against the hydrophilic acidic protein and basic protein was evaluated using sodium dodecyl sulfate–polyacrylamide gel electrophoresis. The successful extraction of BSA from calf serum was also achieved using the resulting monolith as a solid-phase extraction adsorbent. Thus, poly(APTES-MAA-co-DES-co-EDMA) monolith could be a potential material to extract proteins according to their hydrophilicity/hydrophobicity and for further separation based on their acidity/basicity.

Evaluation of mass transfer rate in aqueous two-phase systems: Effect of microchannel width for bovine serum albumin extraction

In this research, extraction of bovine serum albumin (BSA) with aqueous two-phase systems (ATPS) based on polyethylene glycol/sodium citrate using the microfluidic apparatus were investigated. The operational variables were studied using microchannel width (800–400 μm), salt phase flow rate (1.6–4 mL/h), polyethylene glycol-rich phase flow rate (0.8–2 mL/h), and pH (5–9) by response surface methodology (RSM). Double interfaces performed BSA transfer from the salt phase to the polyethylene glycol (PEG) phase. The effects of adding the interface on the mass transfer coefficient and the mass transfer rate were evaluated. Using the microfluidic system due to having two interfaces will increase the extraction rate compared to macro systems with a single interface. In values, salt phase pH of 7, PEG phase flow rate of 2 mL/h, and salt phase flow rate of 4 mL/h were the optimum condition for BSA extraction rate (70.9%.) in this study.

Structural effects of choline amino acid ionic liquids on the extraction of bovine serum albumin by green and biocompatible aqueous biphasic systems composed of polypropylene glycol400 and choline amino acid ionic liquids

Ionic liquid-based aqueous biphasic systems (IL-based ABSs) are considered as promising extraction and purification routes. For designing an efficient process of protein extraction, it is necessary to complete information about phase diagrams including binodal and tie-lines for new ABSs. In this respect, here, binodal curves and tie-lines for ternary aqueous solutions composed of polypropylene glycol 400 and some of choline amino acid ionic liquids, [Ch][AA]-ILs, including choline L-histidine, choline L-arginine, choline L-proline, and choline L-valine as a green and biocompatible entrainers have been determined at T = 298.15 under atmospheric pressure (≈85 kPa). The results indicated that the binodals of these ABSs affected strongly by the ILs structures; so that, biphasic region is increased by decreasing of hydrophobicity of IL anion from valine to arginine. The effective exclude volume theory (EEV) and Setschenow equation have been utilized for addressing phase formation capability in these systems. Phase formation ability in these ABSs followed in the order: choline L-arginine > choline L-histidine > choline L-proline > choline L-valine which is parallel with an increasing hydrophilicity of amino acid exists in each of IL structures. Moreover, osmotic virial equation was used for modeling of tie-lines in the studied ABSs. Finally, the performance of these ABSs for separation of biomolecules was evaluated by studying the partition behavior of bovine serum albumin, BSA, via measuring of partition coefficient and extraction efficiency. The obtained results from BSA partitioning in these ABSs indicated that BSA is migrated preferentially to the [Ch][AA]-ILs-rich bottom phase and this migration is increased by increasing hydrophilicity of amino acid exists in IL structure, i.e. choline L-arginine > choline L-histidine > choline L-proline > choline L-valine. Also, it was found that the salting-out effect and π-π interactions between BSA and [Ch][AA]-ILs have important role in BSA partitioning as well as hydrophilicity/hydrophobicity interactions.

The role of ionic association of choline amino acid ionic liquids on the two-phase formation and extraction of bovine serum albumin in ATPSs containing PEGDME250 and choline histidine or choline arginine at different temperatures

The choline amino acid ionic liquids, [Ch][AA]-ILs, as atoxic task-specific solvents are used for inducing an aqueous two-phase systems, ATPSs, in the presence of polymers such as polyethylene glycol di-methyl ether 250, PEGDME250. In these ILs, depending on anions structure the varied and strong interactions are existed between choline cation, [Ch]+, and amino acid anions, [AA], leading to an increase of ion association. These interactions affect ILs hydration behavior and therefore the studying of these ionic associations in some detail are useful in regard to phase formation ability. In this respect, binodal curve locations and tie-lines for ATPSs composed of PEGDME250 and choline histidine or choline arginine have been determined at three temperatures (298.15–318.15) K under atmospheric pressure (≈85 kPa) and followed by searching the effects of structure and ionic association of ILs together with temperatures on the phase diagram. The ionic association constants of the above ILs together with the previously studied ILs were calculated from ionic conductivity measurements made on the these binary aqueous IL solutions at T = (288.15–318.15) K using low concentration Chemical Model method. For correlation of equilibrium data the Othmar-Tobias, Bancraft and Setschenow equations along with osmotic virial and NRTL (e-or m-NRTL) models were utilized. Finally, influences of ionic association of ILs and temperature were investigated on the separation of bovine serum albumin. The results show that phase forming ability of different [Ch][AA]-ILs is increased by decreasing of Ka values which is parallel with decreasing of amino acid hydrophobicity.

Effect of H-bonding effect on reverse micelle extraction of bovine serum albumin

The extraction of bovine serum albumin (BSA) was carried out by using reverse micelles formed by cationic surfactant dodecyltrimethylammonium bromide (DTAB), N-dodecyl-N-2-hydroxyethyl-N,N-dimethyl-ammonium bromide (HDAB) and N-dodecyl-N,N-2-dihydroxyethyl-N-methyl ammonium bromide (DHAB). The factors like pH, surfactant concentration, salt concentration, and co-solvent concentration were investigated. The good maintenance of the secondary structure of BSA after extraction was confirmed by UV spectra. A wider aqueous phase pH range was achieved and less amount of surfactant was needed on efficient forward extraction when hydroxyethyl group was presented in the surfactants’ head group, suggesting an important role of H-bonding effect. At lower pH or high salt concentration, reasonable backward extraction efficiencies were observed in all systems. The little difference of the head group of these surfactants resulted in quite different performance in the extraction process, which provided deeper insight on the effect of fine adjustment of surfactant structure on protein extraction.

Dithiocarbamate Modified SPION-Chitosan Nanobiocomposite, a Promising Adsorbent for Bovine Serum Albumin (BSA)

In the present paper, bovine serum albumin (BSA) was studied for extraction case from a buffer solution through a new version of modified magnetic chitosan nanocomposite. Post-modification of this magnetic chitosan led to conversion of amine groups to dithiocarbamate on the surface of chitosan which was wrapped to superparamagnetic iron oxide nanoparticles (SPION). Chitosan was converted to magnetic chitosan over co-precipitation of Fe2+ and Fe3+ under alkali conditions. Amines of chitosan were also converted to dithiocarbamate over the reaction of carbon disulfide. Study of the synthesized support in BSA extraction was achieved in further experiments.

Reverse micellar partitioning of Bovine Serum Albumin with novel system

To overcome the difficulties associated with the conventional extraction process like poor selective extrac- tion of biomolecule and scale up of the process, the reverse micellar system consist of AOT/n-heptanol was considered to extract Bovine Serum Albumin (BSA) as a model biomolecule. The maximum forward extraction of BSA from aqueous phase to micelle phase was observed at AOT concentration 160 mM, aque- ous phase pH value of 4, NaCl concentration 0.8 M and 95% back extraction of BSA from micelle phase to stripping phase was obtained at 1 M NaCl concentration with the pH of 7.5. HPLC analysis confirmed the stability of BSA during extraction. The size and water content of the reverse micelle was also reported. The obtained results emphasize the application of the AOT/n-heptanol reverse micellar system for the extraction of BSA and may be utilized for the selective extraction of similar hydrophilic proteins from the complex sources.

Reverse micellar partitioning of Bovine Serum Albumin with novel system

Abstract To overcome the difficulties associated with the conventional extraction process like poor selective extraction of biomolecule and scale up of the process, the reverse micellar system consist of AOT/n-heptanol was considered to extract Bovine Serum Albumin (BSA) as a model biomolecule. The maximum forward extraction of BSA from aqueous phase to micelle phase was observed at AOT concentration 160 mM, aqueous phase pH value of 4, NaCl concentration 0.8 M and 95% back extraction of BSA from micelle phase to stripping phase was obtained at 1 M NaCl concentration with the pH of 7.5. HPLC analysis confirmed the stability of BSA during extraction. The size and water content of the reverse micelle was also reported. The obtained results emphasize the application of the AOT/n-heptanol reverse micellar system for the extraction of BSA and may be utilized for the selective extraction of similar hydrophilic proteins from the complex sources.

Extraction of bovine serum albumin with reverse micelles from glucosylammonium and lactosylammonium surfactants

Abstract Reverse micelle extraction is still in the stage of laboratory. Major limitation associated with use of synthetic surfactants in reverse micelle extraction process is the unfolding or denaturation of proteins. Sugar surfactants are thought non-toxic and environmentally benign, and can exhibit interesting interfacial properties, but the application of sugar-based surfactants in protein extraction is still limited. In the present study, we extracted bovine serum albumin (BSA) by using reverse micelles from glucosylammonium ( GA ) and lactosylammonium ( LA ) surfactants (with dicarboxylate as counter ion). It was found that under optimum condition, (1) the maximum forward extraction efficiency was ca. 86% with GA , while only around 50% with LA , and (2) almost all BSA solubilized in reverse micelles prepared from GA could be recovered into aqueous phase, while the recovery of BSA from the reverse micelles of LA was lower. In addition, the optimum extraction parameters were closely related to surfactant structure. Therefore, the electrostatic interaction, H-bonding and sugar head size should be important for BSA transfer.

Unbound monomers do diffuse through the dentin barrier

ObjectivesAssessing the role of dentinal fluid proteins in trans-dentinal diffusion of free monomers in vitro. MethodsAn artificial pulp chamber (APC) topped human dentin disks was used. A simplified two-step etch-and-rinse adhesive was formulated with 2-hydroethyl-methacrylate (HEMA), Bisphenol-A-diglycidyl-methacrylate (BisGMA), using Camphorquinone/tertiary amine as initiators. Two extraction media were used: buffered saline (Control), buffered saline with 1% bovine serum albumin (BSA). Samples were acid-etched, rinsed, air dried. Simplified primer was used, adhesive applied then light cured with a LED curing. Monomer diffusion was assessed by reverse phase HPLC. ResultsQuantifiable amounts of HEMA were detected in both extraction media while BisGMA was present in quantifiable amounts in BSA medium only. Diffused monomers concentrations were significantly higher for both monomers in BSA extraction medium. SignificanceAlbumin is sometimes referred to as taxi protein for its ability to bind and transport hydrophobic ligands. From our results, we hypothesized that albumin can also transport unbound monomers released from dental adhesive through the dentin barrier. However, dentinal fluid proteins like albumin could have significant effect on monomer diffusion through dentin to the dental pulp transporting highly hydrophobic molecules like BisGMA and enhancing diffusion of more hydrophilic ones like HEMA. These results demonstrate a new possible mechanism for cytotoxicity of resin monomers.

A facile synthesis of novel three-dimensional magnetic imprinted polymers for rapid extraction of bovine serum albumin in bovine calf serum.

A novel three-dimensional (3D) magnetic imprinted polymer was prepared with bovine serum albumin as the template molecule, dopamine as the functional monomer, and a graphene oxide and carbon nanotube hybrid 3D nanocomposite as the carrier. The preparation conditions for the 3D magnetic molecularly imprinted polymers were optimized. The 3D magnetic molecularly imprinted polymers were characterized in detail by scanning electron microscopy, Fourier transform infrared spectroscopy, and vibrating sample magnetometry. The 3D magnetic molecularly imprinted polymers possessed a fast adsorption rate and excellent adsorption performance toward bovine serum albumin, with a maximum adsorption capacity of 78.12mgg-1. The extraction conditions, including the washing solvent, the pH of the eluent, and the desorption time, were also optimized. Combined with high-performance liquid chromatography, the 3D magnetic molecularly imprinted polymers were successfully applied to enrich and separate bovine serum albumin from bovine calf serum samples with recoveries of 84.0-94.5%. Graphical Abstract ᅟ.

Good's Buffer Ionic Liquids as Relevant Phase-Forming Components of Self-Buffered Aqueous Biphasic Systems.

A series of new self-buffering ionic liquids (ILs) based on Good's buffers (GBs) anions and the tetrabutylphosphonium cation ([P4444]+) was here synthesized and characterized. The self-buffering behaviour of the GB-ILs was confirmed by measuring their protonation constants by potentiometry. Further, their ability to form aqueous biphasic systems with the biodegradable potassium citrate salt was evaluated, and further investigated for the extraction of proteins, using bovine serum albumin (BSA) as a model protein. If these ionic structures display self-buffering characteristics as well as a low toxicity towards the luminescent bacteria Vibrio fischeri, they were additionally found to be highly effective in the formation of ABS and in the extraction of BSA - extraction efficiencies of 100% to the IL-rich phase obtained in a single-step. The BSA secondary structure in the aqueous IL-rich solutions was evaluated through infrared spectroscopic studies revealing the protein-friendly nature of the synthesized ILs. Dynamic light scattering (DLS), "COnductor-like Screening MOdel for Real Solvents" (COSMO-RS), and molecular docking studies were finally carried out to better understand the main driving forces of the extraction process. The results suggest that van der Waals and hydrogen-bonding interactions are important driving forces of the protein migration towards the GB-IL-rich phase, while the molecular docking investigations demonstrated a stabilizing effect of the studied ILs over the protein.

Enhanced extraction of proteins using cholinium‐based ionic liquids as phase‐forming components of aqueous biphasic systems

Aqueous biphasic systems (ABS) composed of ionic liquids (ILs) are promising platforms for the extraction and purification of proteins. In this work, a series of alternative and biocompatible ABS composed of cholinium-based ILs and polypropylene glycol were investigated. The respective ternary phase diagrams, tie-lines, tie-line lengths and critical points were determined at 25°C. The extraction performance of these systems for commercial bovine serum albumin (BSA) was then evaluated. The stability of BSA at the IL-rich phase was ascertained by size exclusion high-performance liquid chromatography and Fourier transform infrared spectroscopy. Appropriate ILs lead to the complete extraction of BSA for the IL-rich phase, in a single step, while maintaining the protein's native conformation. Furthermore, to evaluate the performance of these systems when applied to real matrices, the extraction of BSA from bovine serum was additionally carried out, revealing that the complete extraction of BSA was maintained and achieved in a single step. The remarkable extraction efficiencies obtained are far superior to those observed with typical polymer-based ABS. Therefore, the proposed ABS may be envisaged as a more effective and biocompatible approach for the separation and purification of other value-added proteins.

Enhanced extraction of bovine serum albumin with aqueous biphasic systems of phosphonium- and ammonium-based ionic liquids

Novel aqueous biphasic systems (ABS) composed of phosphonium- or ammonium-based ionic liquids (ILs), combined with a buffered aqueous solution of potassium citrate/citric acid (pH=7.0), were investigated for the extraction of proteins. For that purpose, the phase diagrams, tie-lines and tie-line lengths were determined at 25°C, and the performance of these ABS for the extraction of bovine serum albumin (BSA) was then evaluated. The obtained results reveal that, with the exception of the more hydrophobic ILs, most of the systems investigated allow the complete extraction of BSA for the IL-rich phase in a single-step. These remarkable extraction efficiencies are far superior to those afforded by more conventional extraction systems previously reported. The composition of the biphasic systems, i.e., the amount of phase-forming components, was also investigated aiming at reducing the overall costs of the process without losing efficiency on the protein extraction. It is shown that the extraction efficiencies of BSA are maintained at 100% up to high protein concentrations (at least up to 10gL−1). The recovery of the BSA from the IL-rich phase by dialysis is also shown in addition to the demonstration of the IL recyclability and reusability, at least for 3 times. In the sequential three-step extractions (BSA recovery/IL reusability), the extraction efficiencies of BSA for the IL-rich phase were maintained at 100%. For the improved ABS, the preservation of the protein native conformation was confirmed by Size Exclusion High-Performance Liquid Chromatography (used also as the quantification method) and by Fourier Transform Infra-Red spectroscopy. According to the results herein reported, ABS composed of phosphonium- or ammonium-based ILs and a biodegradable organic salt represent an alternative and remarkable platform for the extraction of BSA and may be extended to other proteins of interest.

Deep eutectic solvents as novel extraction media for protein partitioning

Four kinds of green deep eutectic solvent (DES) were synthesized, including choline chloride (ChCl)-urea, tetramethylammonium chloride (TMACl)-urea, tetrapropylammonium bromide (TPMBr)-urea and ChCl-methylurea. An aqueous two-phase system (ATPS) based ChCl-urea DES was studied for the first time for the extraction of bovine serum albumin (BSA). Single factor experiments proved that the extraction efficiency of BSA was influenced by the mass of the DES, concentration of K2HPO4 solution, separation time and extraction temperature. The optimum conditions were determined through an orthogonal experiment with the four factors described above. The results showed that under the optimum conditions, the average extraction efficiency could reach up to 99.94%, 99.72%, 100.05% and 100.05% (each measured three times). The relative standard deviations (RSD) of extraction efficiencies in precision, repeatability and stability experiments were 0.5533% (n = 5), 0.8306% (n = 5) and 0.9829% (n = 5), respectively. UV-vis and FT-IR spectra confirmed that there were no chemical interactions between BSA and the DES in the extraction process, and the CD spectra proved that the conformation of BSA did not change after extraction. The conductivity, DLS and TEM were combined to investigate the microstructure of the top phase and the possible mechanism for the extraction. The results showed that hydrophobic interactions, hydrogen bonding interactions and the salting-out effect played important roles in the transfer process, and the aggregation and surrounding phenomenon were the main driving forces for the separation. All of these results proved that ionic liquid (IL)-based ATPSs could potentially be substituted with DES-based ATPSs to offer new possibilities in the extraction of proteins.

Aqueous two-phase extraction for bovine serum albumin (BSA) with co-laminar flow in a simple coaxial capillary microfluidic device

Microfluidic extraction based on a co-laminar flow of aqueous two-phase system is used to separate bovine serum albumin (BSA). Mass transfer between the continuous two-phase flows is demonstrated by the extraction of BSA in a microfluidic device. The protein concentrations of the BSA samples were determined using the Bradford method. Polyethylene glycol 4000 and ammonium sulfate ((NH4)2SO4) served as model aqueous two-phase solutions. The appropriate flow rates of the aqueous two phases were thus determined. We can flexibly control the mass transfer area and time by simply adjusting the flow rate. It takes only 3.6 s for three extraction cycles in a coaxial microfluidic device to achieve a BSA recovery yield of 71.1 %, which is superior to the traditional beaker aqueous two-phase extraction process. In this study, co-laminar flow-based continuous microextraction is demonstrated and its mass transfer is analyzed by solving the diffusion model, based on a large specific interfacial area and surface renewal.

Modelling and numerical simulation of liquid–solid circulating fluidized bed system for protein purification

A novel liquid–solid circulating fluidized bed (LSCFB) was modelled for protein recovery from the feed broth. A typical LSCFB system consists of downer and riser, integrating two different operations simultaneously. A general purpose, extensible, and dynamic model was written based on the tanks-in-series framework. The model allowed adjusting the degree of backmixing in each phase for both columns. The model was validated with previously published data on extraction of bovine serum albumin (BSA) as model protein. Detailed dynamic analysis was performed on the protein recovery operation. The interaction between the riser and downer were captured. Parametric studies on protein recovery in LSCFB system were carried out using the validated model to better understand the system behaviour. Simulation results have shown that both production rate and overall recovery increased with solids circulation rate, superficial liquid velocity in the downer and riser, and feed solution concentration. The model was flexible and could use various forms of ion exchange kinetics and could simulate different hydrodynamic behaviours. It was useful to gain insight into protein recovery processes. The general nature of the model made it useful to study other protein recovery operations for plant and animal proteins. It could also be useful for further multi-objective optimization studies to optimize the LSCFB system.

Choline-like ionic liquid-based aqueous two-phase extraction of selected proteins

In this paper, an ionic liquid-based aqueous two-phase system (ATPs) has been developed for the effective extraction of bovine serum albumin (BSA). Compared with the effects of three different ionic liquids used in an aqueous two-phase system, a choline-like ionic liquid was selected as the suitable solution for bovine serum albumin. Extraction efficiencies of BSA were found to be dependant on the temperature, time, IL mass and the salt content. Based on single-factor experiments, these significant operating parameters of the ILs-ATP were respectively optimized. Experimental results show that 92.03–100.03% of the proteins could be extracted into the ionic liquid-rich phase in a single-step extraction. Repeatability, precision and stability experiments were investigated and the RSDs are lower than 2.3%, 1.7% and 4.7% respectively. Experiments indicated that intermolecular forces, the micelle phenomenon and the salting-out effect play important roles in the transfer process, and the aggregation phenomenon was the main driving force for the separation. Conformation of the protein was not affected after extraction into the upper ionic liquid-rich phase according to the determination by UV-visible (UV-vis), fluorescence spectra, dynamic light scattering (DLS), Fourier transform infrared (FT-IR) spectroscopy and TEM. It is suggested that the method of choline-like ILs-based ATPs for the selective separation of protein would have potential applications in bio-analysis and bio-separation.