Combined hydrophobic-metal binding fusion tags for applications in aqueous two-phase partitioning.
In this work, we studied the influence of fusion affinity tags containing both hydrophobic and histidines residues on the partitioning of the green fluorescent protein, GFPuv, in aqueous two-phase system. The tags were fused to the N-terminal of GFPuv and tested by immobilized metal affinity partitioning, in a PEG/salt system. The presence of both types of residues in the tag increased the partitioning greatly. Particularly, four engineered tags (H6, FH6, WH6, and YH6) containing a hexa-histidine sequence as well as different hydrophobic residues, all increased partitioning more than twice, reaching K values around 20, as compared to another construct (His6-GFP) containing an isolated hexa-histidine sequence. YH6, also proved be beneficial for protein expression.
Bernaudat
Florent
F
Department of Pure and Applied Biochemistry, Centre for Chemistry and Chemical Engineering, Lund University, P.O. Box 124, 221 00 Lund, Sweden. florent.bernaudat@tbiokem.lth.se
Bülow
Leif
L
D002846:Chromatography, Affinity / Q000379:methods; D006639:Histidine / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification*; D057927:Hydrophobic and Hydrophilic Interactions; D008670:Metals / Q000737:chemistry; D009842:Oligopeptides / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification*; D015202:Protein Engineering / Q000379:methods; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification*
C471213:His-His-His-His-His-His; D008670:Metals; D009842:Oligopeptides; D011993:Recombinant Fusion Proteins; D006639:Histidine