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.
Department of Biochemistry and Biotechnology, Royal Institute of Technology, Stockholm, Sweden.
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K
Köhler
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B
Nilsson
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A
Veide
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