Bio/Technology
9, 642 - 646 (1991)
doi:10.1038/nbt0791-642
Engineering Proteins to Enhance their Partition Coefficients in Aqueous Two-Phase SystemsKristina Köhler1, Charlotta Ljungquist1, Akihiko Kondo1, 2, Andres Veide1, *
& Björn Nilsson1, 3, *
1Department of Biochemistry and Biotechnology, Royal Institute of Technology, S-100 44 Stockholm, Sweden.
2Department of Applied Chemistry, Kyushu Institute of Technology, Sensuicho, Tobata, Kitakyushu 804, Japan.
3Department of Biochemistry, Kabi Pharmacia KabiGen S-112 87 Stockholm, Sweden.
*Corresponding authors. We describe a novel method to partition recombinant proteins into the polymerrich top phase in poly(ethylene glycol) (PEG)4000/potassium phosphate aqueous two-phase systems. The concept is based on fusion of a gene fragment encoding a short peptide sequence to the product gene of interest thereby changing the partitioning properties of the expressed product protein as a fusion to the peptide. The model protein in this study, ZZ, is a two domain molecule based on staphylococcal protein A (SPA) which distributes evenly in PEG/salt systems. A tetrapeptide sequence, AlaTrpTrpPro (designated the partitioning peptide), was designed by molecular modeling techniques to include exposed tryptophan residues and to have a coding DNA sequence which is possible to polymerize in an obligate head-to-tail fashion at the DNA level. Gene fragments encoding one and three partitioning peptides, respectively, were fused to the 3' end of the ZZ gene and the fusion proteins were produced intracellularly in Escherichia coli. The partition coefficients of ZZ proteins containing zero, one and three fused partitioning peptides were determined in three PEG 4000/potassium phosphate aqueous two-phase systems of different compositions. In all three phase systems, there were dramatic effects on the partition coefficient by the fused partitioning peptides. In the phase system with the largest effects, the partition coefficient was enhanced from 1.6 to 11.6 by fusing one tetrapeptide sequence to the 147 amino acid model ZZ protein. By the fusion of three partitioning peptides, the coefficient was increased to 96. These results show that fused partitioning peptides have a dramatic effect on the partitioning of a protein in aqueous two-phase systems which may open up new possibilities to utilize aqueous two-phase extraction techniques as a primary recovery step of recombinant proteins.
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