Secretion of the extracellular domain of the human insulin receptor from insect cells by use of a baculovirus vector

Abstract

To explore the utility of the baculovirus/insect-cell system for the expression of a soluble secreted human insulin-receptor (hIR) extracellular ligand-binding domain, we have engineered a recombinant virus encoding an hIR deletion mutant which is truncated eight residues from the beginning of the predicted transmembrane domain (i.e. 921 residues). Within 24 h after infection of Sf9 cells with virus, insulin-binding activity begins to accumulate in the culture medium, and reaches a maximum between 48 and 72 h. The intracellular transit and processing of this secreted receptor, designated 'AchIR01', is quite slow. After 24 h in pulse-chase experiments approximately 50% of the metabolically labelled protein is still inside the cell. This protein accumulates as a non-cleaved hIR precursor which is glycosylated, but the carbohydrate is entirely endoglycosidase H (endoH)-sensitive (i.e. high mannose). Approximately one-half of the receptor in the culture medium (i.e. approximately 25% of the total) is in the form of non-cleaved precursor, and about one half of its carbohydrate chains are now endoH-resistant. The remainder of the protein is proteolytically processed hIR (alpha-plus truncated beta-subunits). None of these hIR species exhibit O-linked carbohydrate. Only the processed form of the receptor in the medium binds insulin. This insulin-binding protein is secreted as a dimer (alpha beta)2, and binds insulin with an affinity which is comparable with that of both the wild-type hIR as well as the secreted form of the hIR expressed in mammalian cells. Despite the rather inefficient processing and altered glycosylation of the AchIR01 protein in insect cells, this high-affinity insulin-binding protein accumulates in the medium at levels (mg/litre) of about 100 times that achieved in a mammalian-cell system.

Identification of the ligand-binding regions in the macrophage colony-stimulating factor receptor extracellular domain.

“…3 indicate that the secretion of the Fms E protein began later than 24 h postinfection and gradually accumulated in the medium during the next 24 to 48 h. After reaching the maximum at 96 h postinfection, the production of Fms E sharply decreased, coincident with cell lysis. The synthesis and secretion characteristics of Fms E protein are similar to those reported for other proteins, such as epidermal growth factor receptor, insulin receptor, and other receptors (12,43,54 Fig. 4B that N-glycosidase F could remove more carbohydrate groups from the Fms E protein than endoglycosidase H and about half the population of Fms E molecules acquired endoglycosidase H resistance.…”

Overexpression, purification, and characterization of full-length and mutant caldesmons using a baculovirus expression system

“…Synthesis of the full-length CaD was detected 24 h postinfection, and maximal protein accumulation was observed at 36 h postinfection. The time course of PvlCaD synthesis is similar to that of the human c-myc protein (Miyamoto et al,I985), and quite different from those of epidermal growth factor receptor (Greenfield et al, 1988), insulin receptor (Sissom & Ellis, 1989), and los protein (Tratner et al, 1990).…”

Fusion of Insulin Receptor Ectodomains to Immunoglobulin Constant Domains Reproduces High-affinity Insulin Binding in Vitro

“…HIRs-Fc was secreted only after complete cleavage of the proreceptor. In contrast, the truncated receptor produced in baculovirus-transfected Sf9 cells was heterogeneous, with culture medium containing both cleaved and uncleaved receptor forms (30,31). Furthermore, HIRs expressed in both NIH 3T3 cells and Sf9 cells was degraded during chase times between 6 and 24 h after …”