Efficient expression in insect cells of a soluble, active human insulin receptor protein-tyrosine kinase domain by use of a baculovirus vector

Abstract

The human insulin receptor (IR) is a transmembrane glycoprotein, whose cytoplasmic domain contains an insulin-activated protein-tyrosine kinase (EC 2.7.2.112). By the use of an appropriately engineered baculovirus expression vector, a soluble cytoplasmic derivative of this domain was expressed in the insect cell line Spodoptera frugiperda (Sf9). At 24 to 48 h after Sf9 cells were infected with recombinant virus, a protein of the size expected for this domain (-48 kilodaltons) constituted a major band when total cell lysates of metabolically labeled cells were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and fluorography. This protein (designated AchIRPTK) was immunoprecipitated by three monoclonal antibodies, each of which recognizes a distinct antigenic site of the IR cytoplasmic domain and requires the native structure of the protein for recognition and one of which binds at or near the physiologically relevant site(s) of IR autophosphorylation. In vivo, AchIRPTK was phosphorylated on both tyrosine and serine residues. When affinity purified, the kinase was active in vitro; it autophosphorylated exclusively on tyrosine residues, and phosphorylated the exogenous substrates histone H2b and poly(Glu-Tyr). The expression of an active IR protein-tyrosine kinase molecule in this heterologous cell system provides an efficient experimental method for producing this domain in quantity for enzymatic and structural studies.

Phosphorylation Effects on Flanking Charged Residues

“…The engineering of a recombinant baculovirus for the -35-kDa kinase (hIR residues 978-1283) expression in Sf9 cells will be described elsewhere (Wei, L., Hubbard, S., Hendrickson, W., and Ellis, L., unpublished results). Kinase production was basically as described previously for the -48-kDa derivative (hIR residues 959-1355; Ellis et al, 1988;Cobb et al, 1989), except that the infection period was 72-90 h, and the cell lysate was passed down a DE-52 (Whatman) anion-exchange column to remove an abundant, high-molecular-mass protein contaminant before FF' LC. The =35-kDa kinase was eluted from the DE-52 column by 0.15 M NaC1, pH 7.6.…”

Truncation of the ectodomain of the human insulin receptor results in secretion of a soluble insulin binding protein from transfected CHO cells

“…(ii) Are the extracellular and cytoplasmic domains of the IR, in the absence of the other domain, capable of independent function? The cytoplasmic PTK domain of the IR has now been expressed as a soluble protein in the cytoplasm of both rodent (CHO (Ellis et al, 1987a)) and insect (Sf9) cells (Ellis et al, 1988). When purified from such cells, this kinase is recognized by a panel of monoclonal antibodies which require the native conformation of the molecule for binding (Morgan and Roth, 1986).…”

Stable plasma membrane expression of the soluble domain of the human insulin receptor in yeast

“…The soluble cytoplasmic protein tyrosine kinase domain of hInsR has previously been expressed in the baculovirus expression system (Sf9 insect cells) in active form. These studies were undertaken in order to identify autophosphorylated tyrosine and serine/threonine residues [18,36] in InsR (in the absence of insulin) and analyze their importance in tyrosine kinase activity and signal transduction [14–17,37]. Here, we describe the stable expression of the complete cytoplasmic protein fragment of hInsR including the juxtamembrane domain in yeast cells.…”

“…From this failure and from the striking difference in molecular mass and phosphorylation behavior to vertebrate insulin receptors it is evident that, if yeast has an insulin recognizing protein, its primary structure is poorly conserved. We examined whether the soluble cytoplasmic domain of the β‐chain of hIns receptor (hInsR), which has been demonstrated to exhibit similar catalytic properties and to use the identical sites in autophosphorylation as the native holo‐receptor [14–18], could couple to intracellular components of the yeast glucose signalling cascade. We reasoned that these downstream constituents might be conserved in evolution to a higher degree than the putative insulin‐like ligand and its receptor.…”