Purification and characterization of human recombinant insulin-like growth factor binding protein 3 expressed in Chinese hamster ovary cells.
Recombinant human insulin-like growth factor binding protein 3 (hIGFBP-3) stably expressed in chinese hamster ovary cells (CHO cells) has been purified to homogeneity from serum-free culture media. The purified protein migrates as a doublet (45/43 kDa) upon SDS-PAGE. The purified recombinant hIGFBP-3 is fully active and binds one mole of IGF-I per mole of recombinant binding protein. When the transfected CHO cells are treated with tunicamycin a single 29 kDa hIGFBP-3 protein is observed. This expressed hIGFBP-3 protein maintains its ability to bind IGF-I. N-Glycanase treatment of the purified hIGFBP-3 protein results in a protein that migrates similar to E. coli-derived IGFBP-3 upon SDS-PAGE under reducing conditions (30 kDa). Carboxymethylation of hIGFBP-3 suggests that all 18 cysteines are involved in disulfide linkages. These results represent the first purification and characterization of recombinant hIGFBP-3 expressed in CHO cells.
Tressel
T J
TJ
BioGrowth, Inc., Richmond, CA 94806.
Tatsuno
G P
GP
Spratt
K
K
Sommer
A
A
D000818:Animals; D002352:Carrier Proteins / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D002460:Cell Line; D002846:Chromatography, Affinity; D002851:Chromatography, High Pressure Liquid; D006224:Cricetinae; D003412:Cricetulus; D004591:Electrophoresis, Polyacrylamide Gel; D005260:Female; D006801:Humans; D018969:Insulin-Like Growth Factor Binding Proteins; D007334:Insulin-Like Growth Factor I / Q000378:metabolism*; D008970:Molecular Weight; D010053:Ovary; D011994:Recombinant Proteins / Q000302:isolation & purification / Q000378:metabolism; D014162:Transfection
D002352:Carrier Proteins; D018969:Insulin-Like Growth Factor Binding Proteins; D011994:Recombinant Proteins; D007334:Insulin-Like Growth Factor I