Purification of the IL-2 receptor (TAC) by ligand-affinity chromatography and utilization of the immobilized receptor for receptor-affinity chromatography (RAC) purification of IL-2, mutant IL-2, and IL-2 fusion proteins.
Recombinant technology has facilitated the production of two soluble forms of human p55 interleukin-2 receptor (IL-2R) in Chinese hamster ovary cells. We have developed a ligand-affinity method for the medium-scale purification of these two soluble forms of the IL-2R, based on the biochemical interactions between the matrix-bound ligand (interleukin-2) and its soluble receptor. The affinity-purified IL-2R is further purified by anion-exchange chromatography followed by gel filtration. This method has provided enough highly pure IL-2R for structure and function studies and for use in practical applications such as high-flux drug-screening assays. The purified IL-2R subsequently has been immobilized on silica gel and employed for the purification of recombinant IL-2. Receptor-affinity-chromatography-purified IL-2 contains only a highly active monomeric form of the lymphokine, in contrast to immunoaffinity chromatography where several molecular forms of IL-2 with varying degrees of biologic activity are recovered. Receptor-affinity chromatography has been successfully applied to the purification of several mutant IL-2 as well as an IL-2-Pseudomonas exotoxin (IL2-PE40) fusion protein that is a 54.5-kDa chimeric protein in which the cell recognition domain is replaced by IL-2. The IL-2-PE40 is a potential cytotoxic agent for cells bearing the IL-2 receptor.
Smart
J E
JE
Department of Protein Biochemistry, Hoffmann-La Roche Inc., Nutley, New Jersey 07110.
Familletti
P C
PC
Weber
D V
DV
Keeney
R F
RF
Bailon
P
P
D036002:ADP Ribose Transferases; D000818:Animals; D001427:Bacterial Toxins; D002846:Chromatography, Affinity / Q000379:methods; D002850:Chromatography, Gel; D006224:Cricetinae; D003412:Cricetulus; D005098:Exotoxins / Q000032:analysis; D005260:Female; D007376:Interleukin-2 / Q000235:genetics / Q000302:isolation & purification*; D009154:Mutation; D010053:Ovary / Q000032:analysis; D015375:Receptors, Interleukin-2 / Q000302:isolation & purification*; D011993:Recombinant Fusion Proteins / Q000032:analysis*; D037521:Virulence Factors; D000097668:Pseudomonas aeruginosa Exotoxin A
D036002:ADP Ribose Transferases; D001427:Bacterial Toxins; D005098:Exotoxins; D007376:Interleukin-2; D015375:Receptors, Interleukin-2; D011993:Recombinant Fusion Proteins; D037521:Virulence Factors; D000097668:Pseudomonas aeruginosa Exotoxin A