GENETICALLY-ENGINEERED CHARGE MODIFICATIONS TO ENHANCE PROTEIN SEPARATION IN AQUEOUS 2-PHASE SYSTEMS - CHARGE DIRECTED PARTITIONING

This report continues our examination of the effect of genetically eng ineered charge modifications on the partitioning behavior of proteins; in aqueous two-phase extraction. The genetic modifications consisted of the fusion of charged peptide tails to beta-galactosidase and charg e-change point mutations to T4 lysozyme. Our previous article examined the influence of these charge modifications on partitioning as a func tion of interfacial potential difference. In this study, we examined c harge 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 attr active forces between the protein and polymer were present. Nearly 100 % of the beta-galactosidase, which carries a net negative charge, part itioned to the DEAE-dextran-rich phase regardless of whether the phase was dextran or PEG. In these cases, cloudiness of the protein-rich ph ases suggest that strong charge interactions resulted in protein/polym er aggregation, which may have contributed to the extreme partitioning . Unlike the potential-driven partitioning reported previously, consis tent 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-base d interactions. Similarly, T4 lysozyme, carrying a net positive charge , partitioned to the dextran sulfate-containing phase, and displayed f our- to sevenfold shifts in K-p as a result of the point mutations. Th ese shifts were two to four times stronger than those observed for pot ential driven partitioning. Little effect on partitioning was observed when the protein and polymer had the same charge, with the exception of beta-galactosidase with polyarginine tails. The high positive charg e density of these tails provided for a localized interaction with the dextran sulfate, and resulted in 2- to 15-fold shifts in K-p. (C) 199 5 John Wiley and Sons, Inc.