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Two DNA fragments encoding PDZ domain(21-110 residues) and BAR domain(150-360 residues) from PICK1(1-416 residues) were amplified by PCR and then introduced into vectors, pET-32M and pMAL-c2X respectively to generate recombinant plasmids, pE-pdz and pM-bar. Having been separately transferred into the hosts E.coli BL21 and E.coli JM109, these two strains can express fusion proteins: His-tagged PDZ(PDZ domain) and maltose binding protein-BAR(MBP-BAR domain) respectively, as confirmed by both SDS-PAGE and Western blotting. The interaction between these two domains is dose-dependence, as identified by a pull-down test. Moreover, it has been shown from the ELISA analysis that the actual amount of PDZ bound to MBP-BAR-amylose beads reaches (16±0.5)%, as calculated by the molar ratio of PDZ to MBP-BAR. In addition, the interaction between BAR(bait) and PDZ(prey) in vivo was also examined with a yeast two-hybrid system.
Two DNA fragments encoding PDZ domain (21-110 residues) and BAR domain (150-360 residues) from PICK1 (1-416 residues) were amplified by PCR and then introduced into vectors, pET-32M and pMAL-c2X respectively to generate recombinant plasmids, pE-pdz and pM-bar. Having been separately transferred into the hosts E. coli BL21 and E. coli JM109, these two strains can express fusion proteins: His-tagged PDZ (PDZ domain) and maltose binding protein-BAR MBP-BAR domain) respectively, as confirmed by both SDS-PAGE and Western blotting. The interaction between these two domains is dose-dependence, identified as a pull-down test. Moreover, it has been shown from the ELISA analysis that the The actual amount of PDZ bound to MBP-BAR-amylose beads reaches (16 ± 0.5)% as calculated by the molar ratio of PDZ to MBP-BAR. In addition, the interaction between BAR (bait) and PDZ was also examined with a yeast two-hybrid system.