Cloning and Expression of Insulin Receptor Ligand-binding Domains.
Insulin receptor is a transmembrane protein consisting of four subunits, that form a heterotetramer(alpha(2)beta(2))with molecular weight of 350 kD. Because the extracellular subunit(alpha)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 alpha 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.
Zhang
Hong
H
State Key Laboratory of Molecular Biology, Shanghai Institute of Biochemistry, Shanghai Institutes for Biological Sciences, the Chinese Academy of Sciences, Shanghai 200031, China. fengym@sunm.shcnc.ac.cn
Qiao
Zhi-Song
ZS
Feng
You-Min
YM