Matchmaker two hybrid system 2

Yeast two-hybrid screening was performed using the MATCHMAKER Two-Hybrid System 2 (Clontech) [40 (link)–42 (link)]. YRG-2 yeast host cells were purchased from Stratagene. pAS2-1 and pACT2 plasmids were cotransfected and selected on G2 plates deficient in tryptophan and leucine and on G3 plates deficient in histidine. The yeast host cells were MATa ura3–52 his3–200 ade2–101 lys2–801 trp1–901 leu2–3 112 gal4–542 gal80–538 LYS2::UASGAL1-TATA GAL1-HIS3 URA3::UASGAL4 17mers(x3)-TATACYC1-lacZ. A visible blue-color pattern in the colony filter lift assay on the G3 plates represented a positive interaction [41 (link)]. YRG-2 yeast cells were cotransfected with pACT2-GSKIP and an empty pAS2-1 vector and spread on G2 and G3 agar plates to determine the growth-inhibiting effect of GSKIP in yeast.

The plasmids of pACT2-GSKIP and pAS2-1-GSK3β were constructed for the yeast two-hybrid assay as described previously [40 (link)–42 (link)]. Briefly, GSK3β was cloned in-frame with the Gal4 DNA-binding domain in the pAS2-1 vector (MATCHMAKER Two-HybridSystem 2, Clontech) to yield the pAS2-1-GSK3β bait plasmid. In addition, DNA fragments encoding GSKIP were amplified through PCR using Taq polymerase (TaKaRa). The PCR fragments were then inserted into the BamHI and XhoI sites of the pACT2 (Clontech) vector to construct the pACT2-GSKIP plasmid. GSKIP Y118P, Y118A, Y118F, F122P, F122A, F122Y, L126P, F126A, L126V, L126Q, L130P, L130A, L130I, and L130V mutants were created through a site-directed mutagenesis technique by using the QuikChange Lightning kit (GE Healthcare, Sunnyvale, CA, USA). Mutated nucleotides were verified using an ABI PRISM 3730 Genetic Analyzer (Perkin-Elmer) for DNA sequencing. All experimental procedures were performed in accordance with the manufacturer’s protocol.

The Matchmaker two-hybrid system 2 was used according to the protocol recommended by the manufacturer (Clontech Laboratories, Palo Alto, CA, USA). The full-length Haspin cDNA was constructed into a pAS2-1 vector (Clontech Laboratories, Palo Alto, CA, USA) and was transformed into competent yeast Y190 cells. The yeast expressed the recombinant protein of the GAL4-DNA binding domain fusion, and HASPIN was obtained as the tryptophan non-requiring strain. The mouse testis cDNA library was prepared in pACT2, and a vector expressed the fusion protein of the GAL4 transcription-activation domain (Clontech Laboratories, Palo Alto, CA, USA). The pACT2 included in the cDNA library was transformed into tryptophan non-requiring yeast and screened for genes expressing proteins interacting with HASPIN. Positive clones were obtained as tryptophan, leucine, and histidine non-requiring, and were found to express LacZ.