Purification of recombinant proteins from mammalian cell culture using a generic double-affinity chromatography scheme.
Transient transfection of mammalian cells has proven to be a useful technique for the rapid production of recombinant proteins because of its ability to produce milligram quantities within 2 weeks following cloning of their corresponding cDNA. This rapid production also requires a fast and efficient purification scheme that can be applied generically, typically through the use of affinity tags such as the polyhistidine-tag for capture by immobilized metal-affinity chromatography (IMAC) or the Strep-tag II, which binds to the StrepTactin affinity ligand. However, one-step purification using either of these tags has disadvantages in terms of yield, elution conditions, and purity. Here, we show that the addition of both Strep-tag-II and (His)(8) to the C-terminal of r-proteins allows efficient purification by consecutive IMAC and StrepTactin affinity. This approach has been successfully demonstrated using the intracellular protein DsRed, as well as two secreted proteins, secreted alkaline phosphatase (SEAP) and vascular endothelial growth factor (VEGF), all produced by transient transfection of HEK293-EBNA1 cells in medium supplemented with bovine calf serum. All proteins were purified to >99% homogeneity with yields varying from 29 to 81%.
Cass
Brian
B
Animal Cell Technology Group, Bioprocess Sector, Biotechnology Research Institute, National Research Council Canada, 6100 Royalmount Avenue, Montreal, Que., Canada H4P 2R2.
Pham
Phuong Lan
PL
Kamen
Amine
A
Durocher
Yves
Y
D000469:Alkaline Phosphatase / Q000096:biosynthesis / Q000235:genetics / Q000302:isolation & purification; D000595:Amino Acid Sequence; D001483:Base Sequence; D002478:Cells, Cultured; D002846:Chromatography, Affinity / Q000379:methods*; D005822:Genetic Vectors; D006801:Humans; D008164:Luminescent Proteins / Q000096:biosynthesis / Q000235:genetics / Q000302:isolation & purification; D008969:Molecular Sequence Data; D010957:Plasmids / Q000235:genetics; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics / Q000302:isolation & purification*; D014162:Transfection; D042461:Vascular Endothelial Growth Factor A / Q000096:biosynthesis / Q000235:genetics / Q000302:isolation & purification; D000097573:Red Fluorescent Protein
D000469:Alkaline Phosphatase; D008164:Luminescent Proteins; D011994:Recombinant Proteins; D042461:Vascular Endothelial Growth Factor A; D000097573:Red Fluorescent Protein