Matchmaker gal4 yeast two hybrid system

The long carboxyl terminus (CT_L) of rat Ca_v1.3_L variant (GenBank accession no. NM_001389225; Addgene plasmid no. 49332; Xu and Lipscomb, 2001) was amplified by PCR (polymerase chain reaction) and then cloned into bait pGBKT7 vector using NdeI and BamHI sites. CT_L–pGBKT7 construct and rat brain cDNA library in pACT2 vector (Clontech) were co-transformed to AH109 competent cells to isolate novel proteins interacting with CT_L using Matchmaker GAL4 yeast two-hybrid system (Clontech). A positive colony was selected on a high-stringent SD medium deficient of Leu, Trp, His, and Ade, in the presence of X-α-Gal. The vector insert in the positive clone was identified as including the full-length cDNA of rat Snapin2 (GenBank accession no. NM_001025648). The Snapin2 cDNA was subcloned into pcDNA3 and pGEM-HEA vectors for co-expression studies in HEK293-cells and Xenopus oocytes, respectively.
The N-terminus (NT), I-II loop, II-III loop, and III-IV loop of Ca_v1.3_L and the short CT (CT_S) of rat Ca_v1.3_S variant (Addgene plasmid number 49,333; GenBank accession no. AF370009; [10 (link)]) were also amplified by PCR and individually inserted into pGBKT7 bait vector. Their interaction with rat Snapin2 was tested by Y2H assays.

We used the Matchmaker GAL4 yeast two-hybrid system (Clontech, Mountain View, CA, USA) as described in the product manual. PR₅₀ was cloned into the pGBKT7 vector (containing the GAL4 DNA-binding domain DNA-BD) as bait and pre-transformed into the S. cerevisiae host strain of AH109. A cDNA library (Clontech) of the human brain was constructed on the pGADT7 vector (containing the GAL4 activation domain AD) and expressed in the host strain of Y187 as prey. The Y187 library strain was then mated with the PR₅₀-expressing AH109 strain to generate diploids. Transcription of ADE2, HIS3, MEL1, and LacZ reporter genes is activated if DNA-BD and AD in diploids interact via bait and prey fusion proteins to form intact GAL4 transcription factors. This is reflected in blue positive diploids on SD/-Ade/-His/-Leu/-Trp/X-α-gal plates. To identify the cDNA fragments of the prey contained in these positive diploids, we used the Zymoprep™ Yeast Plasmid Miniprep I (Zymo Research Corporation, Irvine, CA, USA) kit to recover prey plasmids from positive diploids on SD/-Ade/-His/-Leu cultures. The plasmids were then sequenced and analyzed.