Genetic engineering of protein-peptide fusions for control of protein partitioning in thermoseparating aqueous two-phase systems

Abstract

Genetic engineering has been used for the fusion of peptides, with different length and composition, on a protein to study the effect on partitioning in aqueous two‐phase systems containing thermoseparating polymers. Peptides containing 2–6 tryptophan residues or tryptophan plus 1–3 lysine or aspartate residues, were fused near the C‐terminus of the recombinant protein ZZT0, where Z is a synthetic IgG‐binding domain derived from domain B in staphylococcal protein A. The partitioning behavior of the peptides and fusion proteins were studied in an aqueous two‐phase system composed of dextran and the thermoseparating ethylene oxide–propylene oxide random copolymer, EO30PO70. The zwitterionic compound β‐alanine was used to reduce the charge‐dependent salt effects on partitioning, and to evaluate the contribution to the partition coefficient from the amino acid residues, Trp, Lys, and Asp, respectively. Trp was found to direct the fusion proteins to the EO–PO copolymer phase, while Asp and Lys directed them to the dextran phase. The effect of sodium perchlorate and triethylammonium phosphate on the partitioning of the fusion proteins was also studied. Salt effects were directly proportional to the net charge of the fusion proteins. Sodium perchlorate was found to be 3.5 times more effective in directing positively charged proteins to the EO–PO copolymer phase compared to the effect of triethyl ammonium phosphate on negatively charged proteins. An empirical correlation has been tested where the fusion protein partitioning is a result of independent contributions from unmodified protein, fused peptide, and salt effects. A good agreement with experimental data was obtained which indicates the possibility, by independent measurements of partitioning of target protein and fusion peptide, to approximately predict the fusion protein partitioning. © 1999 John Wiley & Sons, Inc. Biotechnol Bioeng 62: 135–144, 1999.

Role of alcohols in the formation of inverse microemulsions and back extraction of proteins/enzymes in a reverse micellar system

“…This problem has been solved to some extent using thermoseparating polymers; a suitable way for the recovery of polyethylene glycol (an extensively used polymer in ATPE) is not yet developed [4].…”

Aqueous two-phase systems containing urea: Influence of protein structure on protein partitioning

“…These two contributions are related however, because the hydrophobic residues in proteins have the largest surface of all amino acids. These findings will improve the knowledge base for a wide range of emerging fields like refolding in a phase system (Forciniti, 1994;Spears et al, 1995) or the construction of fusion tags to enhance partitioning (Berggren et al, 1999;Malmsten et al, 1998). Further investigations on charge-modified T4-lysozymes (Luther et al, 1994(Luther et al, , 1995Fan et al, 1998) will give us more insight into the so-called "electrostatic potential" of these systems (Grossman et al, 1997(Grossman et al, , 1998Tintinger et al, 1997aTintinger et al, , 1997b.…”

N-Terminal tagged lactate dehydrogenase proteins: evaluation of relative hydrophobicity by hydrophobic interaction chromatography and aqueous two-phase system partition

“…Partition experiments were performed in ATPS composed of 7.1% (w/w) dextran and 6.8% (w/w) EO30PO70, whose phase diagram has been previously published [18]. The top phase polymer EO30PO70, with a molecular mass of 3300, was obtained from Shearwater Polymers Inc. (Nektar Therapeutics, Huntsville, USA).…”