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.
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.
Persson
J
J
Department of Biochemistry, Lund University, P.O. Box 124, S-221 0 Lund, Sweden.
Nyström
L
L
Ageland
H
H
Tjerneld
F
F
D016632:Apolipoprotein A-I / Q000302:isolation & purification*; D001709:Biotechnology / Q000379:methods*; D004269:DNA, Bacterial / Q000737:chemistry; D004852:Epoxy Compounds / Q000737:chemistry; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D005027:Ethylene Oxide / Q000737:chemistry; D005285:Fermentation; D006801:Humans; D008823:Micelles; D017830:Octoxynol / Q000737:chemistry; D011108:Polymers / Q000737:chemistry; D011136:Polysorbates / Q000737:chemistry; D011994:Recombinant Proteins / Q000302:isolation & purification*; D013501:Surface-Active Agents / Q000737:chemistry; D013696:Temperature
D016632:Apolipoprotein A-I; D004269:DNA, Bacterial; D004852:Epoxy Compounds; D008823:Micelles; D011108:Polymers; D011136:Polysorbates; D011994:Recombinant Proteins; D013501:Surface-Active Agents; D017830:Octoxynol; D005027:Ethylene Oxide; C009068:propylene oxide