Partitioning and characterization of tyrosine-tagged green fluorescent proteins in aqueous two-phase systems.
The green fluorescent protein GFPuv has been genetically engineered to investigate the influence of N-terminal tyrosine extensions in aqueous two-phase systems. Fusions in the N-terminus affected the protein expression, and tags containing three tyrosines and prolines influenced the expression favorably. This effect is probably due to changes in mRNA stability, because the amounts of corresponding mRNAs correlated with the amounts of GFPuv proteins. The partitioning was investigated in two different aqueous two-phase systems, a two-polymer system composed of EO30PO70/dextran and a PEG/salt system with potassium phosphate. Partitioning in the PEG/salt system generally was more favorable than in the EO30PO70/dextran system. Tags with three tyrosines resulted in higher partitioning toward the EO30PO70- and PEG-rich phases, respectively. The effect of adding proline residues to the tag was also investigated, and the partitioning effect of the tag was enhanced when prolines were included in the tags with three tyrosines. The best tyrosine tag, Y3P2, increased the partition coefficient 5 times in the PEG/salt system. Thermoseparation of the EO30PO70 phase allowed recovery of 83% Y3P2-GFPuv protein in a water phase.
Fexby
Sara
S
Department of Pure and Applied Biochemistry, Center for Chemistry and Chemical Engineering, Lund University, PO Box 124, SE-221 00 Lund, Sweden.
Nilsson
Anna
A
Hambraeus
Gustav
G
Tjerneld
Folke
F
Bülow
Leif
L
D045424:Complex Mixtures / Q000737:chemistry / Q000302:isolation & purification; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D049452:Green Fluorescent Proteins / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification*; D016297:Mutagenesis, Site-Directed; D044367:Phase Transition; D015202:Protein Engineering; D011993:Recombinant Fusion Proteins / Q000737:chemistry* / Q000302:isolation & purification*; D012996:Solutions; D013050:Spectrometry, Fluorescence / Q000379:methods*; D014443:Tyrosine / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification*; D014867:Water / Q000737:chemistry
D045424:Complex Mixtures; D011993:Recombinant Fusion Proteins; D012996:Solutions; D014867:Water; D049452:Green Fluorescent Proteins; D014443:Tyrosine