Expression, Purification, and Characterization of the Cytoplasmic Domain of the Human IGF-1 Receptor Using a Baculovirus Expression System

The cytoplasmatic domain of the β-subunit of the human IGF-1 receptor (residues 929–1337) has been overexpressed in insect cells using the baculovirus expression system. Synthesis of the soluble protein (IGFK, Mr 46 kDa) in Spodoptera frugiperda (Sf9) cells was detected 24 h after infection and maximal accumulation was achieved 40–48 h postinfection. Rapid purification to near homogeneity (≥95% pure protein) was accomplished by sequential chromatography on Resource-Q and phenyl-Sepharose with a specific activity of 142 nmol/min/mg using poly[Glu:Tyr] as substrate. The purified IGFK showed a preference for Mn2+ ions and a linear incorporation of 32P from [γ-32P]ATP over a 20-fold dilution of the protein and was stimulated 20-fold by the polycation poly-l-lysine. Interestingly, the kinase autophosphorylated on tyrosine and serine residues. In contrast, a kinase-negative mutant, IGFK-K1003A, did not undergo phosphorylation on tyrosine or serine residues, respectively, suggesting that IGF-1 receptor kinase is a dual specific kinase.