[Screening of AGGF1 interacting with inhibitor of differentiation-1].
OBJECTIVE
To screen the proteins interacting with inhibitor of differentiation 1(Id1) using yeast two-hybrid analysis in adult human lung cDNA libraries.


METHODS
The 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.


RESULTS
Successful 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.


CONCLUSIONS
AGGF1 is confirmed to interact with Id1 by yeast two-hybrid analysis for screening adult human lung cDNA libraries.
Jia
Si-yuan
SY
Department of Burns, Zhujiang Hospital, Southern Medical University, Guangzhou 510282, China.
Luo
Xiang-dong
XD
Qi
Jie
J
D000328:Adult; D042501:Angiogenic Proteins / Q000235:genetics / Q000378:metabolism*; D049109:Cell Proliferation; D042783:Endothelial Cells / Q000166:cytology / Q000378:metabolism; D015723:Gene Library; D006801:Humans; D051798:Inhibitor of Differentiation Protein 1 / Q000378:metabolism*; D008168:Lung / Q000166:cytology; D010957:Plasmids / Q000235:genetics; D011485:Protein Binding; D020798:Two-Hybrid System Techniques
C482200:AGGF1 protein, human; D042501:Angiogenic Proteins; D051798:Inhibitor of Differentiation Protein 1