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Updated: May 15, 2026
Split-Ubiquitin Based Membrane Yeast Two-Hybrid (MYTH) System: A Powerful Tool For Identifying Protein-Protein Interactions
Published on: February 1, 2010
[Construction and expression identification of human secreted apoptosis-related protein 1 gene yeast two-hybrid bait
Wei Zhang1, Guangzhao He, Bing Ma
1Department of Medical Cosmetology, the Affiliated Hospital ofXi'an Medical University, Xi'an Shaanxi, 710077, P.R.China.
Objective:
To construct human secreted apoptosis-related protein 1 (SARP1) gene yeast two-hybrid bait vector so as to study the biological functions of the SARP1 gene in the scar tissue.
Methods:
The target gene from SARP1 gene full-length DNA segment was amplified by PCR, the upstream and downstream primers of the SARP1 gene with restriction enzymes Nde I and Sal I were designed. pGBKT7-SARP1 recombination plasmid was constructed by ligating the vector and the PCR production and identified by PCR and sequencing. Further more, pGBKT7-SARP1 was transformed into competent AH109 which contained kanamycin for selecting positive clones and screened the positive clony on the plate of SD/-Trp. The toxicity and transcriptional activation were tested by the phenotype assay.
Results:
SARP1 was amplified and cloned into pGBKT7 successfully, SARP1 gene sequence in recombinant plasmid pGBKT7-SARP1 was verified by gel electrophoresis and DNA sequencing analysis. The sequence of inserted SARP1 gene was the same as the corresponding sequence found in GenBank database. The recombinant pGBKT7-SARP1 plasmids and empty pGBKT7 vector could form white colonies on SD/-Trp plates and none could survive on SD/-Leu plates.
Conclusion:
The recombinant pGBKT7-SARP1 gene yeast two-hybrid bait vector is successfully constructed.
