Screening of AGGF1 interacting with inhibitor of differentiation-1.
- Author:
Si-yuan JIA
1
;
Xiang-dong LUO
;
Jie QI
Author Information
- Publication Type:Journal Article
- MeSH: Adult; Angiogenic Proteins; genetics; metabolism; Cell Proliferation; Endothelial Cells; cytology; metabolism; Gene Library; Humans; Inhibitor of Differentiation Protein 1; metabolism; Lung; cytology; Plasmids; genetics; Protein Binding; Two-Hybrid System Techniques
- From: Journal of Southern Medical University 2007;27(9):1379-1381
- CountryChina
- Language:Chinese
-
Abstract:
OBJECTIVETo screen the proteins interacting with inhibitor of differentiation 1(Id1) using yeast two-hybrid analysis in adult human lung cDNA libraries.
METHODSThe coding sequence of Id1 was amplified by PCR and cloned into the bait plasmid. The recombinant bait vector pHybLex/Zeo-Id1 was verified by restriction endonuclease digestion before transformation into the yeast strain EGY48/pSH18-34, which was tested subsequently for reporter genes Leu2 and LacZ activation. The pHybLex/Zeo-Id1 plasmid and the cDNA library plasmid were sequentially transformed into the yeast strains and screened to obtain Leu2(+) and Leu2(+)LacZ(+) clones, with the false positive clones excluded using positive and negative controls, and the plasmid of the true positive clone was sequenced and blasted for homological analysis.
RESULTSSuccessful construction of pHybLex/Zeo-Id1 was confirmed by enzyme digestion. After transformation of pHybLex/Zeo-Id1 into EGY48/pSH18-34, no specific reporter genes Leu2 and LacZ activation was found. The pHybLex/Zeo-Id1 plasmid and the cDNA library plasmid were sequentially transformed into yeast strain, and 198 Leu(+) clones and 19 Leu(+)LacZ(+) double positive clones were obtained. After elimination of the false positive clones, one true positive clone was obtained, whose plasmid analysis by sequencing and blasting indicated high homology (99.5%, 556/559) to AGGF1 (an angiogenic factor with G-patch and FHA domains 1). AGGF1 expression was confirmed in the true positive yeast cells by Western blotting.
CONCLUSIONAGGF1 is confirmed to interact with Id1 by yeast two-hybrid analysis for screening adult human lung cDNA libraries.