Another version of the human insulin receptor kinase domain: expression, purification, and characterization.
We have overexpressed another insulin receptor kinase molecule, which consists of residues 941-1343 inclusive of the human insulin receptor, by using the baculo-virus expression vector pVL941. Unlike the two previous preparations of insulin receptor kinase in this expression system, this molecule contains the complete unmodified sequence of the cytoplasmic domain of the human insulin receptor beta subunit. Our construct allows high-level expression of the recombinant protein in cultured Sf9 cells and in cabbage looper (Trichoplusia ni) larvae. An improved purification procedure yields greater than or equal to 95% pure protein in 55% yield from the cells. The specific activity of this purified protein is 3.5-fold greater than from the previously described baculovirus insulin receptor kinase (that included residues 946-1343 from the proreceptor). Like the latter molecule, the insulin receptor kinase molecule reported here sediments as a monomer, and its autophosphorylation occurs by an intramolecular process. Preliminary data about the spectroscopic features of the cytosolic domain of the human insulin receptor are presented.
Villalba
M
M
Program in Molecular Biology, Memorial Sloan-Kettering Cancer Center, New York, NY 10021.
Wente
S R
SR
Russell
D S
DS
Ahn
J C
JC
Reichelderfer
C F
CF
Rosen
O M
OM
D000818:Animals; D001483:Base Sequence; D002460:Cell Line; D015870:Gene Expression; D005796:Genes; D005822:Genetic Vectors; D006801:Humans; D007700:Kinetics; D008969:Molecular Sequence Data; D015345:Oligonucleotide Probes; D010957:Plasmids; D011487:Protein Conformation; D011505:Protein-Tyrosine Kinases / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D011972:Receptor, Insulin; D011994:Recombinant Proteins / Q000302:isolation & purification / Q000378:metabolism; D015183:Restriction Mapping
D015345:Oligonucleotide Probes; D011994:Recombinant Proteins; D011505:Protein-Tyrosine Kinases; D011972:Receptor, Insulin