Engineering proteins to enhance their partition coefficients in aqueous two-phase systems.
We describe a novel method to partition recombinant proteins into the polymer-rich 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.(ABSTRACT TRUNCATED AT 250 WORDS)
Department of Biochemistry and Biotechnology, Royal Institute of Technology, Stockholm, Sweden.
K
K
Köhler
C
C
Ljungquist
A
A
Kondo
A
A
Veide
B
B
Nilsson
D000595:Amino Acid Sequence; D001483:Base Sequence; D003198:Computer Simulation; D008957:Models, Genetic; D008969:Molecular Sequence Data; D010710:Phosphates; D011188:Potassium; D017680:Potassium Compounds; D015202:Protein Engineering / Q000379:methods*; D011994:Recombinant Proteins / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification; D012995:Solubility; D013056:Spectrophotometry, Ultraviolet; D014364:Tryptophan / Q000032:analysis; D014867:Water
D010710:Phosphates; D017680:Potassium Compounds; D011994:Recombinant Proteins; D014867:Water; D014364:Tryptophan; C013216:potassium phosphate; D011188:Potassium