Protein purification via aqueous two-phase extraction (ATPE) and immobilized metal affinity chromatography. Effectiveness of salt addition to enhance selectivity and yield of GFPuv.
This study illustrates the compatibility and complementary nature of aqueous two-phase extraction (ATPE) and immobilized metal affinity chromatography (IMAC) in a general recovery scheme. The purification of green fluorescent protein (GFPuv) from extracts of Eschericia coli was investigated using a combination of these two techniques. High molarity of sodium chloride was found effective in increasing selectivity, with the promotion of hydrophobic interaction the probable mechanism that drove the target protein to a particular phase in ATPE, as well as that which enhanced GFPuv adsorption in IMAC. Moreover, the similar salt condition allows the direct application of the GFPuv-containing phase to the IMAC column without additional adjustment step. A simple screen of conditions was therefore performed to generate a favorable two-step purification scheme for GFP leading to an overall high purity.
D002846:Chromatography, Affinity / Q000379:methods*; D003911:Dextrans / Q000737:chemistry*; D004586:Electrophoresis; D004926:Escherichia coli / Q000145:classification / Q000378:metabolism; D049452:Green Fluorescent Proteins; D008164:Luminescent Proteins / Q000096:biosynthesis / Q000302:isolation & purification*; D008670:Metals / Q000737:chemistry; D011092:Polyethylene Glycols / Q000737:chemistry*; D011506:Proteins / Q000302:isolation & purification; D011786:Quality Control; D012492:Salts / Q000737:chemistry*; D012680:Sensitivity and Specificity; D014867:Water / Q000737:chemistry
D003911:Dextrans; D008164:Luminescent Proteins; D008670:Metals; D011506:Proteins; D012492:Salts; D014867:Water; D049452:Green Fluorescent Proteins; D011092:Polyethylene Glycols