Two methods for improved purification of full-length mammalian proteins that have poor expression and/or solubility using standard Escherichia coli procedures.
Many mammalian proteins are multifunctional proteins with biological activities whose characterization often requires in vitro studies. However, these studies depend on generation of sufficient quantities of recombinant protein and many mammalian proteins cannot be easily expressed and purified as full-length products. One example is the Wilm's tumor gene product, WT1, which has proven difficult to express as a full-length purified recombinant protein using standard approaches. To facilitate expression of full-length WT1 we have developed approaches that optimized its expression and purification in Escherichia coli and mammalian cells. First, using a bicistronic vector system, we successfully expressed and purified WT1 containing a C-terminal tandem affinity tag in 293T cells. Second, using a specific strain of E. coli transformed with a modified GST vector, we successfully expressed and purified N-terminal GST tagged and C-terminal 2x FLAG tagged full-length human WT1. The benefits of these approaches include: (1) two-step affinity purification to allow high quality of protein purification, (2) large soluble tags that can be used for a first affinity purification step, but then conveniently removed with the highly site-specific TEV protease, and (3) the use of non-denaturing purification and elution conditions that are predicted to preserve native protein conformation and function.
Geng
Jinming
J
Renal-Electrolyte and Hypertension Division, Department of Medicine, University of Pennsylvania, Philadelphia, PA 19014, USA.
Carstens
Russ P
RP
D002846:Chromatography, Affinity / Q000379:methods*; D018076:DNA, Complementary / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics* / Q000378:metabolism; D005822:Genetic Vectors; D006801:Humans; D008957:Models, Genetic; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D012995:Solubility; D014162:Transfection; D025721:WT1 Proteins / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism
D018076:DNA, Complementary; D011993:Recombinant Fusion Proteins; D025721:WT1 Proteins