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

We have examined the effect of genetically engineered charge modificat ions on the partitioning behavior of proteins in dextran/polyethylene glycol two-phase systems containing potassium phosphate. By geneticall y altering a protein's charge, the role of charge on partitioning can be assessed directly without the need to modify the phase system. The charge modifications used are of two types: charged tails of polyaspar tic acid fused to beta-galactosidase and charge-change point mutations of T4 lysozyme which replace positive lysine residues with negative g lutamic acids. The partition coefficient K for these proteins was rela ted to measured interfacial potential differences Delta phi using the simple thermodynamic model, ln K-p = ln K-o + (F/RT)Z(p) Delta phi. Th e protein net charge Z(p) was determined using the Henderson-Hasselbal ch relationship with modifications based on experimentally determined titration and isoelectric point data. it was found that when the elect ropartitioning term Z(p) Delta phi was varied by changing the pH, the partitioning of lysozyme was quantitatively described by the thermodyn amic model. The beta-galactosidase fusions displayed qualitative agree ment, and although less than predicted, the partitioning increased mor e than two orders of magnitude for the pH range examined. Changes in t he partitioning of lysozyme due to the various mutations agreed qualit atively with the thermodynamic model, but with a smaller than expected dependence on the estimated charge differences. The beta-galactosidas e fusions, on the other hand, did not display a consistent charge base d trend, which is likely due either to the enzyme's targe size and com plexity or to nonelectrostatic contributions from the tails. The lack of quantitative fit with the model described above suggests that the a ssumptions made in developing this model are oversimplified. (C) 1994 John Wiley & Sons, Inc.