Recombinant protein of the first two ectodomains of cadherin 23 from erl mice shows impairment in Ca2+-dependent proteolysis protection

Abstract

The erl mouse is a mouse model of nonsyndromic autosomal recessive deafness (DFNB12) on the C57BL/6J background. This project was carried out to express the first two ectodomains of cadherin 23 (CDH23 EC1+2) of erl mice in Escherichia coli and identify the Ca2+-binding ability of the recombinant protein. DNA sequences of CDH23 EC1+2 from wild type and erl mice were synthesized and cloned into pBV220 plasmids. Recombinant plasmids were transformed into Escherichia coli and expression of CDH23 EC1+2 was induced by increasing the temperature from 30 °C to 42 °C. The proteins were analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and antigenicity of proteins was identified by Western Blotting. Inclusion bodies were denatured in 8 M urea, purified by ion-exchange and gel filtration chromatography and refolded with dialysis in buffer containing 0.1% sarkosyl. The Ca2+-binding ability of CDH23 EC1+2 was determined by Ca2+-dependent proteolysis protection. The results showed that the sizes and sequences of inserts in recombinant plasmids were consistent with expectation and that the recombinant proteins were found mainly in the form of inclusion bodies which maintain antigenicity. After refolding, the secondary structures of recombinant proteins were measured by circular dichroism (CD) spectra. Moreover, CDH23 EC1+2 from the erl mice showed less Ca2+-dependent proteolysis protection comparing with that of the wild type control. We therefore concluded that impairment of Ca2+-dependent protein interaction was likely involved in the progressive hearing loss in erl mice. The results may aid in understanding the mechanism of hearing loss in DFNB12.