Expression, purification, and characterization of the cytoplasmic domain of the human IGF-1 receptor using a baculovirus expression system.
The cytoplasmatic domain of the beta-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, M(r) 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 Mn(2+) ions and a linear incorporation of (32)P from [gamma-(32)P]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.
Institute of Biochemistry, University of Cologne, Otto-Fischer-Strasse 12-14, Cologne, D-50674, Germany.
N
N
Tennagels
C
C
Hube-Magg
A
A
Wirth
V
V
Noelle
H W
HW
Klein
D000255:Adenosine Triphosphate / Q000378:metabolism; D019943:Amino Acid Substitution; D000818:Animals; D016367:Baculoviridae / Q000235:genetics; D002460:Cell Line; D003600:Cytosol / Q000201:enzymology / Q000378:metabolism; D006801:Humans; D007700:Kinetics; D008274:Magnesium / Q000494:pharmacology; D008345:Manganese / Q000494:pharmacology; D010446:Peptide Fragments / Q000096:biosynthesis / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D010766:Phosphorylation; D011107:Polylysine / Q000494:pharmacology; D017526:Receptor, IGF Type 1 / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D012694:Serine / Q000378:metabolism; D012995:Solubility; D018411:Spodoptera; D013997:Time Factors; D014443:Tyrosine / Q000378:metabolism
D010446:Peptide Fragments; D011994:Recombinant Proteins; D011107:Polylysine; D014443:Tyrosine; D008345:Manganese; D012694:Serine; D000255:Adenosine Triphosphate; D017526:Receptor, IGF Type 1; D008274:Magnesium