1. Soluble expression of human Id3 in Escherichia coli and generation and application of its polyclonal antibodies.
- Author
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Li, Xiao-Jun, Jia, Li, Chen, Fang-Fang, Zhong, Ai-Fang, Yu, Wei, Wang, Kai, and Luo, Bing
- Subjects
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GENE expression , *BACTERIAL genetics , *ESCHERICHIA coli , *BACTERIAL antibodies , *DNA , *RECOMBINANT proteins , *CELL differentiation , *PLASMID genetics , *BINDING sites - Abstract
Inhibitor of DNA binding differentiation 3 (Id3), 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 Id3 ( hId3) gene was amplified by polymerase chain reaction and inserted into prokaryotic expression vector pET32a(+). The recombinant plasmid pET32a/ hId3 was transformed into Escherichia coli BL21 (DE3). The histidine-Tag-fused protein was expressed by induction of 1 mM isopropylthio-β- d-galactoside and purified by Ni2+-nitrilotriacetic acid-agarose column chromatography. The purified hId3 protein was used to generate rabbit polyclonal antisera that recognize recombinant hId3 (rhId3). The antibody was purified by polypeptide affinity chromatography and used for analysis of Id3 subcellular localization in several kinds of tumor cells by indirect immunofluoresence assay. A large quantity of purified rhId3 protein and polyclonal anti-hId3 antibodies would be useful reagents for the further study of biological functions of hId3. [ABSTRACT FROM AUTHOR]
- Published
- 2011
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