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. 2003 Sep;9(9):1892-6.
doi: 10.3748/wjg.v9.i9.1892.

Using yeast two-hybrid system to identify ECRG2 associated proteins and their possible interactions with ECRG2 gene

Yong-Ping Cui  1 , Jian-Bo Wang, Xin-Yu Zhang, Mei-Xia Bi, Li-Ping Guo, Shih-Hsin Lu
Affiliations

Using yeast two-hybrid system to identify ECRG2 associated proteins and their possible interactions with ECRG2 gene

Yong-Ping Cui et al. World J Gastroenterol. 2003 Sep.

Abstract

Aim: To identify esophageal cancer related gene2 (ECRG2) associated proteins and their possible interactions with ECRG2 gene.

Methods: In the yeast forward two-hybrid system, ECRG2 was fused with the DNA-binding domain (DBD) of Gal4 and human fetal liver cDNA library was fused with the transcriptional activation domain (AD) of Gal4. We performed a high-stringency scale procedure to screen ECRG2 against human fetal liver cDNA library and characterized positives by sequence analysis.

Results: We found the following 9 putatively associated proteins. They were metallothionein2A, metallothionein1H, metallothionein1G, ferritin, erythrocyte membrane protein band4.2, mitochondrial ribosomal protein S12, hypothetical protein FLJ10101, and a novel gene whose cDNA was found to have no strong homology to any other previously characterized gene whose DDBJ/EMBL/GenBank accession number is AF422192 mapped to human chromosome 14q31.

Conclusion: MT, a potential interaction partner for ECRG2, might be involved in the regulation of cell proliferation and apoptosis, and in various physiological processes. Determination of a reliability score for each single protein-protein interaction, especially interaction of ECRG2 and MT, permits the assignment of ECRG2 and unannotated proteins to biological pathways. A further understanding of the association between ECRG2 and MT should facilitate the functions of ECRG2 gene.

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Figures

Figure 1
Figure 1
Colony-lift filter assay for β-galactosidase activity. Left: β-galactosidase activity of AH109 transformed with pGBKT7-ECRG2. Right: β-galactosidase activity of AH109 transformed with pCL1 (positive control). The results showed that pCL1 could activate reporter genes, but pGBKT7-ECRG2 could not.
Figure 2
Figure 2
Positive colonies screened by yeast two-hybrid sys-tem using full-length cDNA of ECRG2 as baits. A: PCR products of positive clones digested with HaeIII restriction enzyme. Lane 1: λDNA/EcoR I+Hind III Marker, Lanes 2-16: positive colonies, B: Trp+/Leu+/His+/Ade+ positive clone growing on the SD/Trp-/Leu-/His-/Ade- plate, C: colony-lift filter assay for β-galactosidase activity.
Figure 3
Figure 3
Blast results of MT2A.
Figure 4
Figure 4
Blast results of AF422192.
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