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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
Author(s) -
Persson Josefine,
Nyström Lena,
Ageland Hans,
Tjerneld Folke
Publication year - 1999
Publication title -
biotechnology and bioengineering
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.136
H-Index - 189
eISSN - 1097-0290
pISSN - 0006-3592
DOI - 10.1002/(sici)1097-0290(19991120)65:4<371::aid-bit1>3.0.co;2-p
Subject(s) - pulmonary surfactant , polymer , lower critical solution temperature , chemistry , phase (matter) , micelle , copolymer , chemical engineering , starch , chromatography , aqueous solution , materials science , polymer chemistry , organic chemistry , biochemistry , engineering
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.