Recovery of extracellular human insulin-like growth factor-I and II as a fusion protein from Escherichia coli culture broth by aqueous two-phase extraction.
The primary purification of human insulin-like growth factor-I (IGF-I) and IGF-II, produced extracellularly in Escherichia coli as a fusion to two domains (ZZ) derived from staphylococcal protein A, has been studied. First, the partitioning of IgG-affinity purified ZZ-IGF-I and ZZ-IGF-II, respectively, to the top phase in poly(ethylene glycol)/potassium phosphate aqueous two-phase systems were investigated. Thereafter, the extraction of ZZ-IGF-I with a poly(ethylene glycol) 1500/potassium phosphate system was performed directly in the bioreactor after the cultivation. This resulted in a reduction of the cultivation volume more than 3-fold with a recovery of about 90% of target protein in a poly(ethylene glycol)-rich phase. The majority of the cells partitioned to the potassium phosphate-rich bottom phase, while a smaller fraction was collected at the interface, and/or as a densely packed cake on top of the interface. Contaminating proteins were also eliminated to some extent, which resulted in an almost 2-fold protein purification. Some obvious benefits offered by the aqueous two-phase system in the primary purification have been demonstrated: Firstly, the possibility to an early process volume reduction and thereby a concentration of the target protein. Secondly, a simultaneous protein purification was achieved. From this work it can be concluded that aqueous two-phase extraction should be considered as an attractive candidate for the primary steps during the design of new purification processes for extracellular proteins.
Köhler
K
K
Department of Biochemistry and Biotechnology, Royal Institute of Technology, Stockholm, Sweden.
Nilsson
B
B
Veide
A
A
D002469:Cell Separation; D002846:Chromatography, Affinity; D002851:Chromatography, High Pressure Liquid; D003470:Culture Media; D004926:Escherichia coli; D005285:Fermentation; D006801:Humans; D007074:Immunoglobulin G / Q000276:immunology; D007334:Insulin-Like Growth Factor I / Q000235:genetics / Q000302:isolation & purification*; D007335:Insulin-Like Growth Factor II / Q000235:genetics / Q000302:isolation & purification*; D011993:Recombinant Fusion Proteins / Q000302:isolation & purification*; D013205:Staphylococcal Protein A / Q000276:immunology
D003470:Culture Media; D007074:Immunoglobulin G; D011993:Recombinant Fusion Proteins; D013205:Staphylococcal Protein A; D007334:Insulin-Like Growth Factor I; D007335:Insulin-Like Growth Factor II