Pas2 1 vector

Yeast two-hybrid screening was performed essentially as previously described (Sherman et al., 2001 (link)). Briefly, a random-primed rat sciatic nerve cDNA library in λACTII was screened using the C-terminal region of rat L-PRX (residues 681–1383) in the pAS2-1 vector (Clontech) as bait. Three independent clones of β4 integrin each containing the third FNIII domain were found. To identify the domain of PRX, which interacted with β4 integrin, deletion constructs of PRX were made by PCR and subcloned into pAS2-1. β-galactosidase activity was tested by filter lift assays with one of the β4 integrin clones (62BpACTII).

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.