Cloning and Expression
of Insulin Receptor Ligand-binding Domains
ZHANG Hong, QIAO Zhi-Song, FENG You-Min*
( State Key Laboratory of Molecular Biology, Shanghai Institute of
Biochemistry, Shanghai Institutes for Biological Sciences, the Chinese Academy
of Sciences, Shanghai 200031, China )
Abstract Insulin
receptor is a transmembrane protein consisting of four subunits, that form a
heterotetramer(α2β2)with molecular weight of ～350 kD. Because the
extracellular subunit(α)consists of 731 residues and a cysteine-rich domain, it
is difficult to express and crystallize such a large ligand-binding subunit,
thus hampering further study on“insulin-receptor”complex. Based on the fact
that the domains L1 and L2 of the α subunit, consisted of 119 and 118 residues,
contained the high and low affinity insulin binding sites, respectively, the
cDNAs of L1 and L2 were obtained from a human placental cDNA library by PCR.
The cDNAs of L1, L2 and L1-(Ala)10-L2(designed ten-alanine-connected
L1 and L2)were cloned, respectively, into an expression plasmid pET-3a, and E.coli
BL21(DE3)transformants with such plasmids were successfully induced to express
the goal proteins. The expression products were isolated and purified by the
washing and solubilization of inclusion body, gel filtration chromatography and
ion exchange chromatography. Each final product displayed a single band,
corresponding the purity above 99%, in SDS-PAGE. These products have also been
confirmed respectively as the L1, L2 and L1-(Ala)10-L2 by DNA
sequencing, amino acid composition analysis and N-terminal amino acid
sequencing.
Key words insulin；receptor；extracellular domain
*Corresponding author：Tel,86-21-64374430；Fax,86-21-64338357；e-mail,[email protected]