Cloning and expression of 6xHis tagged protein fragments of mouse cdh23 and pcdh15 were previously described in [39 (link),41 (link)]. Briefly, DNA sequences coding for both protein fragments were cloned using the NdeI and XhoI sites of the pET21a vector that includes a C-terminal His tag. All mutants used in this work were generated using the QuikChange Lightning mutagenesis kit (Stratagene) and were verified by DNA sequencing. Numbering of residues corresponds to mouse CDH23 and PCDH15 without their signal sequences.
Fragments used in SPR experiments were derived from a template provided by Dr. Yoshie Narui. Briefly, a DNA construct containing the N-terminal amino acid residues 1–205 (EC1+EC2) of mouse CDH23 was obtained by PCR amplification and subcloned using the Impact kit (NEB #E6901S) and the NdeI and SapI restriction sites of the pTXB1 vector (NEB #N6707). The pTXB1 attached the Mycobacterium xenopi GyrA intein tag at the C terminus of EC2. The tag contains an N-terminal cysteine residue that allows thiol-induced cleavage. Each intein tag contains a chitin-binding domain (CBD) for affinity purification of the fusion protein on a chitin resin. Induction of on-column cleavage, using thiol reagents such as dithiothreitol (DTT), releases the "tagless" cdh23 from the intein tag. An Ala residue was included between CDH23 EC2 and the intein tag to improve cleavage efficiency.
Fragments used in SPR experiments were derived from a template provided by Dr. Yoshie Narui. Briefly, a DNA construct containing the N-terminal amino acid residues 1–205 (EC1+EC2) of mouse CDH23 was obtained by PCR amplification and subcloned using the Impact kit (NEB #E6901S) and the NdeI and SapI restriction sites of the pTXB1 vector (NEB #N6707). The pTXB1 attached the Mycobacterium xenopi GyrA intein tag at the C terminus of EC2. The tag contains an N-terminal cysteine residue that allows thiol-induced cleavage. Each intein tag contains a chitin-binding domain (CBD) for affinity purification of the fusion protein on a chitin resin. Induction of on-column cleavage, using thiol reagents such as dithiothreitol (DTT), releases the "tagless" cdh23 from the intein tag. An Ala residue was included between CDH23 EC2 and the intein tag to improve cleavage efficiency.
Free full text: Click here
