Advanced

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

Persson, Josefine; Nyström, Lena; Ageland, Hans and Tjerneld, Folke LU (1999) In Biotechnology and Bioengineering 65(4). p.371-381
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,... (More)
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. (Less)
Please use this url to cite or link to this publication:
author
organization
publishing date
type
Contribution to journal
publication status
published
subject
keywords
downstream processing, apolipoprotein A-1, purification, thermoseparating polymer, surfactant, recycling, aqueous two-phase system
in
Biotechnology and Bioengineering
volume
65
issue
4
pages
371 - 381
publisher
John Wiley & Sons
external identifiers
  • scopus:0033589830
ISSN
1097-0290
language
English
LU publication?
yes
id
c4ad1347-b48e-42f9-b779-d3e8e23d85fc (old id 125377)
alternative location
http://www3.interscience.wiley.com/cgi-bin/abstract/71003550/ABSTRACT
date added to LUP
2007-07-06 13:00:09
date last changed
2017-01-01 05:16:54
@article{c4ad1347-b48e-42f9-b779-d3e8e23d85fc,
  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.},
  author       = {Persson, Josefine and Nyström, Lena and Ageland, Hans and Tjerneld, Folke},
  issn         = {1097-0290},
  keyword      = {downstream processing,apolipoprotein A-1,purification,thermoseparating polymer,surfactant,recycling,aqueous two-phase system},
  language     = {eng},
  number       = {4},
  pages        = {371--381},
  publisher    = {John Wiley & Sons},
  series       = {Biotechnology and Bioengineering},
  title        = {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},
  volume       = {65},
  year         = {1999},
}