| 1. |
- Persson, Josefine, et al.
(författare)
-
Aqueous polymer two-phase systems formed by new thermoseparating polymers
- 2000
-
Ingår i: Bioseparation. - 1573-8272. ; 9:2, s. 105-116
-
Tidskriftsartikel (refereegranskat)abstract
- A set of new polymers that can be used as phase forming components in aqueous two-phase systems is presented. All polymers studied have thermoseparating properties i.e. form one separate polymer enriched phase and one aqueous solution when heated above the critical temperature. This property makes the polymers attractive alternatives to the polymers used in traditional aqueous two-phase systems such as poly(ethylene glycol) (PEG) and dextran. The thermal phase separation simplifies recycling of the polymers, thus making the aqueous two-phase systems more cost efficient and suitable for use in large scale. Thermoseparating polymers studied have been copolymers of ethylene oxide and propylene oxide (EO-PO), poly (N-isopropylacrylamide) (poly-NIPAM), poly vinyl caprolactam (poly-VCL) and copolymers of N-isopropylacrylamide and vinyl caprolactam with vinyl imidazole (poly(NIPAM-VI) and poly(VCL-VI), respectively). In addition, the copolymer poly(NIPAM-VI) has the property to be uncharged at pH above 7.0 and positively charged at lower pH. This allows the partitioning of protein to be directed by changing the pH in the system instead of the traditional addition of salt to direct the partitioning. Hydrophobically modified EO-PO copolymer (HM-(EO-PO)) with alkyl groups (C_{14}) at both ends forms two-phase system with for example poly(NIPAM-VI). The phase diagram for poly(NIPAM-VI)/HM-(EO-PO) was determined and the model proteins lysozyme and BSA were partitioned in this system. For BSA in poly(NIPAM-VI)/HM-(EO-PO) system a change in pH from 8.0 to 5.4 results in a change of partition coefficient from K=0.8 to K=5.1, i.e. BSA could be transferred from the HM-(EO-PO) phase to the poly(NIPAM-VI) phase. BSA partitioning in poly(NIPAM-VI)/HM-(EO-PO) system allows quantitative BSA recovery, and recoveries of poly(NIPAM-VI) and HM-(EO-PO) were 53% and 92%, respectively, after the thermoseparation step.
|
|
| 2. |
|
|
| 3. |
- Johansson, Hans-Olof, et al.
(författare)
-
Thermoseparating water/polymer system: A novel one-polymer aqueous two-phase system for protein purification
- 1999
-
Ingår i: Biotechnology and Bioengineering. - 1097-0290. ; 66:4, s. 247-257
-
Tidskriftsartikel (refereegranskat)abstract
- In this study we show that proteins can be partitioned and separated in a novel aqueous two-phase system composed of only one polymer in water solution. This system represents an attractive alternative to traditional two-phase systems which uses either two polymers (e.g., PEG/dextran) or one polymer in high-salt concentration (e.g., PEG/salt). The polymer in the new system is a linear random copolymer composed of ethylene oxide and propylene oxide groups which has been hydrophobically modified with myristyl groups (C14H29) at both ends (HM-EOPO). This polymer thermoseparates in water, with a cloud point at 14degreesC. The HM-EOPO polymer forms an aqueous two-phase system with a top phase composed of almost 100% water and a bottom phase composed of 5-9% HM-EOPO in water when separated at 17-30degreesC. The copolymer is self-associating and forms micellar-like structures with a CMC at 12 µM (0.01%). The partitioning behavior of three proteins (lysozyme, bovine serum albumin, and apolipoprotein A-1) in water/HM-EOPO two-phase systems has been studied, as well as the effect of various ions, pH, and temperature on protein partitioning. The amphiphilic protein apolipoprotein A-1 was strongly partitioned to the HM-EOPO-rich phase within a broad-temperature range. The partitioning of hydrophobic proteins can be directed with addition of salt. Below the isoelectric point (pI) BSA was partitioned to the HM-EOPO-rich phase and above the pI to the water phase when NaClO4was added to the system. Lysozyme was directed to the HM-EOPO phase with NaClO4, and to the water phase with Na-phosphate. The possibility to direct protein partitioning between water and copolymer phases shows that this system can be used for protein separations. This was tested on purification of apolipoprotein A-1 from human plasma and Escherichia coli extract. Apolipoprotein A-1 could be recovered in the HM-EOPO-rich phase and the majority of contaminating proteins in the water phase. By adding a new water/buffer phase at higher pH and with 100 mM NaClO4, and raising the temperature for separation, the apolipoprotein A-1 could be back-extracted from the HM-EOPO phase into the new water phase. This novel system has a strong potential for use in biotechnical extractions as it uses only one polymer and can be operated at moderate temperatures and salt concentrations and furthermore, the copolymer can be recovered. © 1999 John Wiley & Sons, Inc. Biotechnol Bioeng 66: 247-257, 1999.
|
|
| 4. |
- Persson, Josefine, et al.
(författare)
-
Biomolecule Separation Using Temperature-Induced Phase Separation with Recycling of Phase-Forming Polymers
- 2000
-
Ingår i: Industrial & Engineering Chemistry Research. - : American Chemical Society (ACS). - 0888-5885 .- 1520-5045. ; 39:8, s. 2788-2796
-
Tidskriftsartikel (refereegranskat)abstract
- We review our recent work on the thermoseparating polymers in aqueous two-phase extractions of biomolecules. Random copolymers of ethylene oxide and propylene oxide (EOPO copolymers) are thermoseparating in water solution, i.e., at temperatures above the cloud point a concentrated copolymer phase and a water phase almost free of copolymer are formed. This phase behavior makes it possible to recycle copolymers after biomolecule extraction. A system for protein purification has been developed based on an EOPO copolymer/hydroxypropyl starch aqueous two-phase system. The target protein is extracted into the EOPO phase. Nonionic surfactants have been added to improve the partitioning to the copolymer phase. The EOPO phase is removed after extraction, and the temperature is increased above the cloud point. The target protein is recovered in the water phase, and the copolymer and surfactant is obtained as a concentrated phase free from protein. Copolymer and surfactant can be recovered after thermoseparation to 85-90% and four recycle steps have been accomplished. Covalent binding of an affinity ligand to the EOPO copolymer can improve the partitioning of a target protein to the EOPO phase. The EOPO ligand can be recycled after thermal separation. A novel one-polymer aqueous two-phase system containing only thermoseparating EOPO copolymer and water has been developed for smaller biomolecules, e.g., peptides. Above the copolymer's cloud point, hydrophobic peptides will partition to the EOPO-enriched phase and hydrophilic peptides to the water phase. Proteins can be extracted in a one-polymer phase system containing a hydrophobically modified EOPO copolymer (HM-EOPO). The target protein is partitioned to the HM-EOPO phase. The copolymer can be recycled after back-extraction of the protein to a new water phase.
|
|
| 5. |
- Persson, Josefine, et al.
(författare)
-
Purification of protein and recycling of polymers in a new aqueous two-phase system using two thermoseparating polymers
- 1999
-
Ingår i: Journal of Chromatography A. - 0021-9673. ; 864:1, s. 31-48
-
Tidskriftsartikel (refereegranskat)abstract
- In this study we present a new aqueous two-phase system where both polymers are thermoseparating. In this system it is possible to recycle both polymers by temperature induced phase separation, which is an improvement of the aqueous two-phase system previously reported where one of the polymers was thermoseparating and the other polymer was dextran or a starch derivative. The polymers used in this work are EO50PO50, a random copolymer of 50% ethylene oxide (EO) and 50% propylene oxide (PO), and a hydrophobically modified random copolymer of EO and PO with aliphatic C14H29-groups coupled to each end of the polymer (HM-EOPO). In water solution both polymers will phase separate above a critical temperature (cloud point for EO50PO50 50oC, HM-EOPO, 14oC) and this will for both polymers lead to formation of an upper water phase and a lower polymer enriched phase. When EO50PO50 and HM-EOPO are mixed in water, the solution will separate in two phases above a certain concentration i.e. an aqueous two-phase system is formed analogous to poly(ethylene glycol) (PEG)/dextran system. The partitioning of three proteins, bovine serum albumin, lysozyme and apolipoprotein A-1, has been studied in the EO50PO50/HM-EOPO system and how the partitioning is affected by salt additions. Protein partitioning is affected by salts in similar way as in traditional PEG/dextran system. Recombinant apolipoprotein A-1 has been purified from a cell free E. coli fermentation solution. Protein concentrations of 20 and 63 mg/ml were used, and the target protein could be concentrated in the HM-EOPO phase with purification factors of 6.6 and 7.3 giving the yields 66 and 45%, respectively. Recycling of both copolymers by thermoseparation was investigated. In protein free systems 73 and 97.5% of the EO50PO50 and HM-EOPO polymer could be recycled respectively. Both polymers were recycled after aqueous two-phase extraction of apolipoprotein A-1 from a cell free E. coli fermentation solution. Apolipoprotein A-1 was extracted to the HM-EOPO phase with contaminating proteins in the EO50PO50 phase. The yield (78%) and purification factor (5.5) of apolipoprotein A-1 was constant during three polymer recyclings. This new phase system based on two thermoseparating polymers is of great interest in large scale extractions where polymer recycling is of increasing importance.
|
|
| 6. |
- Persson, Josefine, et al.
(författare)
-
Purification of recombinant and human apolipoprotein A-1 using surfactant micelles in aqueous two-phase systems: Recycling of thermoseparating polymer and surfactant with temperature-induced phase separation
- 1999
-
Ingår i: Biotechnology and Bioengineering. - 1097-0290. ; 65:4, s. 371-381
-
Tidskriftsartikel (refereegranskat)abstract
- An effective system has been developed for purification of apolipoprotein A-1 from Escherichia coli fermentation solution and human plasma using aqueous two-phase extraction and thermal-phase separation. The system included non-ionic surfactants (Triton or Tween) and as top phase-forming polymer a random copolymer of ethylene oxide (50%) and propylene oxide (50%), Breox PAG 50A 1000, was used. The bottom phase-forming polymer was either hydroxypropyl starch, Reppal PES 100 and PES 200, or hydroxyethyl starch, Solfarex A 85. The top-phase-forming polymer and the surfactants are thermoseparating in water solution, i.e., when heated a water phase and a polymer/surfactant phase are formed. Recombinant apolipoprotein A-1, the Milano variant, was extracted from E. coli fermentation solution in a primary Breox-starch phase system followed by thermal separation of the Breox phase where the target protein was recovered in the water phase. Both in the Breox-starch system and in the water-Breox system Triton X-100 was partitioned to the Breox phase. The addition of non-ionic surfactants to the Breox-starch system had strong effect on the purification and yield of the amphiphilic apolipoprotein A-1. In a system containing 17% Breox PAG 50A 1000, 12% Reppal PES 100 and addition of 1% Triton X-100 the purification factor was 7.2, and the yield 85% after thermal separation of the Breox phase. Recycling of copolymer and surfactant was possible after thermal separation of copolymer phase. Approximately 85% of the copolymer and surfactant could be recycled in each extraction cycle. DNA could be strongly partitioned to the starch phase in the primary-phase system. This resulted in a 1000-fold reduction of E. coli DNA in the apolipoprotein A-1 solution obtained after thermoseparation. In extraction from human plasma containing low concentrations of apolipoprotein A-1, it was possible to reach a purification factor of 420 with 98% yield. By reducing the volume ratio to 0.1 Apo A-1 could be concentrated in a small volume of top phase (concentration factor 10) with a yield of 85% and a purification factor of 110. © 1999 John Wiley & Sons, Inc. Biotechnol Bioeng 65: 371-381, 1999.
|
|
| 7. |
- Persson, Josefine, et al.
(författare)
-
Purification of recombinant apolipoprotein A-1Milano expressed in Escherichia coli using aqueous two-phase extraction followed by temperature-induced phase separation
- 1998
-
Ingår i: Journal of Chromatography. B. - 1387-2273. ; 711:1-2, s. 97-109
-
Tidskriftsartikel (refereegranskat)abstract
- A method for purification of recombinant apolipoprotein A1 in aqueous two-phase systems has been studied. A mutant of apolipoprotein A-I, the Milano variant, was expressed in E. coli. Phase systems containing ethylene oxide (EO)-propylene oxide (PO) random copolymers have been used. These polymers are thermoseparating and have the ability to separate into one water-rich and one polymer-rich phase when heated above a critical temperature i.e. the cloud point. The filtrate from an E. coli fermentation was added to a primary aqueous two-phase system composed of an EO-PO copolymer and Reppal, which is an inexpensive hydroxypropyl starch. Apolipoprotein A-I was partitioned to the top EO-PO copolymer phase and contaminating proteins to the bottom starch phase. The phase diagrams for Reppal PES 100-EO50PO50 (Ucon) and Reppal PES 100-EO30PO70 were determined. The effect on partitioning, when changing parameters such as polymer concentration, type of polymer, protein concentration, pH, salt concentration and volume ratio, were studied. Studies on E. coli DNA partitioning showed that DNA could be partitioned strongly to the bottom phase. An optimal system was scaled up from 5 g to 5 kg with similar degrees of purification, i.e. 2.5 and 2.7 and yields of 79% and 82% respectively. Furthermore temperature-induced phase formation was used for separation of apolipoprotein A-I from the copolymer by raising the temperature above the copolymer cloud point; thus, recovering protein in a `clean' water phase.
|
|
| 8. |
- Umakoshi, Hiroshi, et al.
(författare)
-
Model process for separation based on unfolding and refolding of chymotrypsin inhibitor 2 in thermoseparating polymer two-phase systems
- 2000
-
Ingår i: Journal of Chromatography. B. - 1387-2273. ; 743:1-2, s. 13-19
-
Tidskriftsartikel (refereegranskat)abstract
- For the design of a new separation process based on unfolding and refolding of protein, the partitioning behaviour of proteins was studied in thermoseparating polymer two-phase systems with varying pH and temperature. Chymotrypsin inhibitor 2 (CI2), which unfolds reversibly in a simple two-state manner, was partitioned in an aqueous two-phase system (ATPS) composed of a random copolymer of ethylene oxide and propylene oxide (Breox) and dextran T-500. Between 25 and 50oC, the partition coefficients of CI2 in Breox-dextran T-500 systems remain constant at neutral pH. However, there is a drastic increase at pH values below 1.7, 2.1, and 2.7 at 25, 40 and 50oC, respectively. The partitioning behavior of CI2 was also investigated in thermoseparating water-Breox systems at 55-60oC, where CI2 was partitioned to the polymer-rich phase at pH values below 2.4. These results on the CI2 partitioning can be explained by the conformational difference between the folded and the unfolded states of the protein, where the unfolded CI2 with a more hydrophobic surface is partitioned to the relatively hydrophobic Breox phase in both systems. A separation process is presented based on the partitioning behavior of unfolded and refolded CI2 by control of pH and temperature in thermoseparating polymer two-phase systems. The target protein can be recovered through (i) selective separation in Breox-dextran systems, (ii) refolding in Breox phase, and (iii) thermoseparation of primary Breox phase.
|
|
