|Title||Expression and purification of human interleukin-2 simplified as a fusion with green fluorescent protein in suspended Sf-9 insect cells.|
|Publication Type||Journal Article|
|Year of Publication||1999|
|Authors||Cha, HJ, Dalal, NG, Vakharia, VN, Bentley, WE|
|Date Published||1999 Mar 26|
|Keywords||Animals, Blotting, Western, Cell Line, Chromatography, Affinity, Electrophoresis, Polyacrylamide Gel, Gene Expression, Genetic Vectors, Green Fluorescent Proteins, Humans, Interleukin-2, Luminescent Proteins, Nucleopolyhedrovirus, Plasmids, Recombinant Fusion Proteins, Spodoptera|
A fusion protein of human interleukin-2 (hIL-2) and green fluorescent protein (GFP) was expressed in insect Sf-9 cells infected with recombinant baculovirus derived from the Autographa californica nuclear polyhedrosis virus (AcNPV). This fusion protein was comprised of a histidine affinity ligand for simplified purification using immobilized metal affinity chromatography (IMAC), UV-optimized GFP (GFPuv) as a marker, an enterokinase cleavage site for recovery of hIL-2 from the fusion, and the model product hIL-2. Successful production of hIL-2 as a fusion protein (approximately 52,000 Da) with GFPuv was obtained. GFPuv enabled rapid monitoring and quantification of the hIL-2 by simply checking the fluorescence, obviating the need for Western blot and/or ELISA assays during infection and production stages. There was no increased 'metabolic burden' due to the presence of GFPuv in the fusion product. The additional histidine residues at the N-terminus enabled efficient one-step purification of the fusion protein using IMAC. Additional advantages of GFP as a fusion marker were seen, particularly during separation and purification in that hIL-2 containing fractions were identified simply by illumination with UV light. Our results demonstrated that GFP was an effective non-invasive on-line marker for the expression and purification of heterologous protein in the suspended insect cell/baculovirus expression system.
|Alternate Journal||J. Biotechnol.|