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Genetically engineered charge modifications to enhance protein separation in aqueous two‐phase systems: Charge directed partitioning
Author(s) -
Luther John R.,
Glatz Charles E.
Publication year - 1995
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/bit.260460109
Subject(s) - dextran , chemistry , lysozyme , peg ratio , aqueous solution , polymer , phase (matter) , charge (physics) , biophysics , charge density , chemical physics , chromatography , chemical engineering , biochemistry , organic chemistry , biology , physics , finance , quantum mechanics , engineering , economics
This report continues or examination of the effect of genetically engineered charge modifications on the partitioning behavior of proteins in aqueous two‐phase extration. The genetic modifications consisted of the fusion of charged peptide tails to β‐galactosidase and charge‐change point mutations to T4 lysozyme. Our previous article examined the influence of these charge modifications on partitioning as a function of interfacial potential difference. In this study, we examined charge directed partitioning behavior in PEG/dextran systems containing small amounts of the charged polymers diethylaminoethyl‐dextran (DEAE‐dextran) or dextran sulfate. The best results were obtained when attractive forces between the protein and polymer were present. Nearly 100% of the β‐galactosidase, which carries a net negative charge, partitioned to the DEAE‐dextran‐rich phase regardless of whether the phase was dextran or PEG. In these cases, cloudiness of the protein‐rich phases suggest that strong charge interactions resulted in protein/polymer aggregation, which may have contributed to the extreme partitioning. Unlike the potentialdriven partitioning reported previously, consistent partitioning trends were observed as a result of the fusion tails, with observed shifts in partition coefficient ( K p ) of up to 37‐fold. However, these changes could not be solely attributed to charge‐based interactions. Similarly, T4 lysozyme, carrying a net positive charge, partitioned to the dextran sulfate‐containing phase, and displayed four‐ to sevenfold shifts in K p as a result of the point mutations. These shifts were two to four times stronger than those observed for potential driven partitioning. Little effect on partitioning was observed when the protein and polymer had the same charge, with the exception of β‐galactosidase with polyarginine tails. The high positive charge density of these tails provided for a localized interaction with the dextran sulfate, and resulted in 2‐ to 15‐fold shifts in K p . © 1995 John Wiley & Sons, Inc.