Genetically engineered charge modifications to enhance protein separation in aqueous two-phase systems: Electrochemical partitioning
- Iowa State Univ., Ames, IA (United States). Dept. of Chemical Engineering
The authors examined the effect of genetically engineered charge modifications on the partitioning behavior of proteins in dextran/polyethylene glycol two-phase systems containing potassium phosphate. By genetically 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 polyaspartic acid fused to [beta]-galactosidase and charge-change point mutations of T4 lysozyme which replace positive lysine residues with negative glutamic acids. The partition coefficient K[sub p] for these proteins was related to measured interfacial potential differences [Delta][phi] using the simple thermodynamic model, In K[sub p] = In K, + (FIRT)Z[sub p] [Delta][phi]. The protein net charge Z[sub p] was determined using the Henderson-Hasselbalch relationship with modifications based on experimentally determined titration and isoelectric point data. It was found that when the electropartitioning term Z[sub p] [Delta][phi] was varied by changing the pH, the partitioning of T4 lysozyme was quantitatively described by the thermodynamic model. The [beta]-galactosidase fusions displayed qualitative agreement, and although less than predicted, the partitioning increased more than two orders of magnitude for the pH range examined. Changes in the partitioning of lysozyme due to the various mutations agreed qualitatively with the thermodynamic model, but with a smaller than expected dependence on the estimated charge differences. The [beta]-galactosidase fusions, on the other hand, did not display a consistent charge based trend, which is likely due either to the enzyme's large size and complexity or to nonelectrostatic contributions from the tails. The lack of quantitative fit with the model described above suggests that the assumptions made in developing this model are oversimplified.
- OSTI ID:
- 7265401
- Journal Information:
- Biotechnology and Bioengineering; (United States), Vol. 44:2; ISSN 0006-3592
- Country of Publication:
- United States
- Language:
- English
Similar Records
Assignment of the backbone sup 1 H and sup 15 N NMR resonances of bacteriophage T4 lysozyme
Lysozyme net charge and ion binding in concentrated aqueous electrolyte solutions