Prokaryotic Expression, Purification, Antibody Production of a NH2-Terminal Fragment of mCLCA3 Protein and Analysis of mClCA3 Protein Expression in Asthmatic Mouse Lung

mCLCA3 is a member of calcium activated chloride channel (CACC) family that may play an important role in mucin packaging and secretion in asthmatic and cystic fibrosis lung. To study the protein structure and expression of mCLCA3 in asthmatic mouse lung, an N-terminal 269 amino acid peptide of mCLCA3 was expressed in E. coli, purified to homogeneity and rabbit polyclonal antibodies against this peptide were generated. Immunohistochemistry of asthmatic mouse lung using the antibody indicated exclusive mCLCA3 expression in mucin granules of goblet cells in airway surface and lumen. Immunoblot analysis of lavage fluid from asthmatic mouse lung revealed a single 90 kDa protein form of mClCA3. The results demonstrate that the 90 kDa N-terminal peptide, neither the full-length protein nor the reported N-terminal 35 kDa cleaved form of mClCA3 is the major functional form involved in the packaging and exocytosis of mucin granules in asthmatic goblet cells.