Purification and properties of insulin receptor ectodomain from large-scale mammalian cell culture.
Ectodomain of the exon 11+ form of the human insulin receptor (hIR) was expressed in the mammalian cell secretion vector pEE6.HCMV-GS, containing the glutamine synthetase gene. Following transfection of the hIR ectodomain gene into Chinese hamster ovary (CHO-K1) cells, clones were isolated by selecting for glutamine synthetase expression with methionine sulphoximine. The expression levels of ectodomain were subsequently increased by gene amplification. Production was scaled up using a 40-liter airlift fermenter in which the transfected CHO-K1 cells were cultured on microcarrier beads, initially in medium containing 10% fetal calf serum (FCS). By continuous perfusion of serum-free medium into the bioreactor, cell viability was maintained during reduction of FCS, which enabled soluble hIR ectodomain to be harvested for at least 22 days. Harvests were concentrated 20-fold by anion-exchange chromatography. Optimal recovery of ectodomain from early harvests containing large quantities of serum proteins was achieved by insulin-affinity chromatography, whereas in later harvests purification was achieved by multistep chromatography. Analysis of the purified hIR ectodomain showed that it had a molecular weight by sedimentation equilibrium analysis of 269,500. Amino-terminal amino acid sequence analysis showed that the ectodomain was correctly processed to alpha and beta chains and that glycosylation characteristics were similar to those of native hIR. The integrity of the ectodomain was demonstrated by the recognition of conformation-dependent anti-hIR antibodies and by its binding of insulin (Kd approximately 2 x 10(-9) M). These results demonstrate the successful production and purification of hIR ectodomain by processes amenable to scale-up and in a form appropriate for structure/function studies of the ligand-binding domain of the receptor.
Cosgrove
L
L
Division of Biomolecular Engineering, CSIRO, Parkville, Victoria, Australia.
Lovrecz
G O
GO
Verkuylen
A
A
Cavaleri
L
L
Black
L A
LA
Bentley
J D
JD
Howlett
G J
GJ
Gray
P P
PP
Ward
C W
CW
McKern
N M
NM
D000818:Animals; D001709:Biotechnology; D016466:CHO Cells; D003001:Cloning, Molecular; D006224:Cricetinae; D005091:Exons; D015870:Gene Expression; D005822:Genetic Vectors; D006801:Humans; D007328:Insulin / Q000378:metabolism; D007700:Kinetics; D008970:Molecular Weight; D011487:Protein Conformation; D011972:Receptor, Insulin / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification*; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification; D014162:Transfection
D007328:Insulin; D011994:Recombinant Proteins; D011972:Receptor, Insulin