Partitioning of beta-galactosidase fusion proteins in PEG/potassium phosphate aqueous two-phase systems.
Four different beta-galactosidase fusion proteins have been partitioned in poly(ethylene glycol) (PEG) 4000/potassium phosphate aqueous two-phase systems. The partition coefficients (K) of staphylococcal protein A-beta-galactosidase (SpA beta gal) (K = 3.5) and staphylococcal protein A-streptococcal protein G-beta-galactosidase (AG beta gal) (K = 2.8) were compared with the partition coefficients of their constituent molecules, beta-galactosidase, SpA, and protein AG. It was found that by fusing beta-galactosidase to the smaller proteins SpA and protein AG, their partition coefficients were increased four to five times. Experimental data were fitted into, and found to agree with, the Albertsson partition model of interacting molecules. The compatibility with PEG and potassium phosphate of beta-galactosidase, SpA, and two different versions of the SpA beta gal protein, displayed as precipitation curves, showed a relationship to the protein partition coefficients in PEG/potassium phosphate systems. High solubility in one phase component was accompanied by preferential partitioning to the phase rich in the same component in the PEG/potassium phosphate system. Also, a changed linker region in SpA beta gal resulted in a more soluble protein. This, together with the improved K values of the target proteins by fusion, shows that it is possible to use beta-galactosidase as an affinity handle.
Department of Biochemistry and Biotechnology, Royal Institute of Technology, Stockholm, Sweden.
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Köhler
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Veide
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Enfors
D003001:Cloning, Molecular / Q000379:methods; D004926:Escherichia coli / Q000235:genetics; D010710:Phosphates; D010957:Plasmids; D011092:Polyethylene Glycols; D011188:Potassium; D017680:Potassium Compounds; D011993:Recombinant Fusion Proteins / Q000302:isolation & purification; D001616:beta-Galactosidase / Q000235:genetics / Q000302:isolation & purification*
D010710:Phosphates; D017680:Potassium Compounds; D011993:Recombinant Fusion Proteins; D011092:Polyethylene Glycols; C013216:potassium phosphate; D001616:beta-Galactosidase; D011188:Potassium