Permeation associated with three-phase-partitioning method on release of green fluorescent protein

Transformed cells of Escherichia coli expressing recombinant green fluorescent protein (GFPuv) were subjected to two methods of extraction: (1) freezing/thawing/sonication (FTS) cycles prior to the three-phase partitioning (TPP) method, or (2) directly to TPP extraction. The amount of GFPuv released by the FTS plus TPP method varied: 374µg/mL (first cycle), 93–442 µg/mL (second cycle), 32–359 µg/mL (third cycle), 18–115 µg/mL (fourth cycle). The GFPuv yields by the second method (TPP only) were, 23–54 µg/mL for the first extract and 33–91 µg/mL for the second. The FTS plus TPP method released similar amounts of GFPuv to that extracted by TPP; and provided a better mixture elution through the hydrophobic interaction column: 13–63 µg/mL for FTS plus TPP methods, and 2.5–13 µg/mL for TPP. The results showed that although selective permeation is a more laborious methodology, it was more efficient for obtaining of GFPuv in relation to the direct extraction of the cells for TPP.