Soluble expression of human Id3 in Escherichia coli and generation and application of its polyclonal antibodies

Inhibitor of DNA binding differentiation (3) (Id(3)), a member of the Id helix loop helix protein family, plays important roles in cell differentiation, cell cycle control, and apoptosis. In the present study, the human Id(3) (hld(3)) gene was amplified by polymerase chain reaction and inserted into prokaryotic expression vector pET(3)2a(+). The recombinant plasmid pET(3)2a/hld(3) was transformed into Escherichia coli BL21 (DE3). The histidine Tag-fused protein was expressed by induction of 1 mM isopropylthio-beta-D-galactoside and purified by Ni2+ nitrilotriacetic acid agarose column chromatography. The purified hld(3) protein was used to generate rabbit polyclonal antisera that recognize recombinant h1d(3) (rhld(3)). The antibody was purified by polypeptide affinity chromatography and used for analysis of Id(3) subcellular localization in several kinds of tumor cells by indirect immunofluoresence assay. A large quantity of purified rhld(3) protein and polyclonal anti-h1d(3) antibodies would be useful reagents for the further study of biological functions of hld(3).(C) 2011 International Union of Biochemistry and Molecular Biology, Inc. Volume 58, Number 2, March/April 2011, Pages 91-96. E-mail: lixiaojun62@yahoo.com.cn