Expression, mutagenesis, and characterization of the cytoplasmic domain and preliminary analysis of ansh2 domain in the membrane-spanning domain of erythrocyte band 3

The thesis is concerned with a family of close related, integral membrane proteins of ubiquitous occurrence: the band 3 proteins. Its structure features a molecule with two relatively independent functional domains. The C-terminal hydrophobic domain (MSDB3) functions as an anion transporter, while the N-terminal hydrophilic domain (CDB3) extends into the cytosol and carries binding sites for cytosolic and cytoskeletal proteins. Using a novel biotinylated primer based site-directed mutagenesis protocol, we have cloned, expressed and purified both a functional dimeric erythrocyte CDB3 and a N-terminal truncated kidney isoform of CDB3 in E.coli. The overexpressed recombinant proteins were isolated in pure form using two simple chromatography steps. Cloned erythrocyte CDB3 exhibits the same structural and biological functions as natured CDB3 except the cloned protein displays slightly weaker binding affinity for glycolytic enzymes due to the fact its N-terminus is not acetylated. However, the N-terminal 65 amino acid truncated kidney CDB3 displayed no binding affinity for ankyrin, protein 4.1 and glycolytic enzymes although it retained most structural properties of erythrocyte CDB3. In other studies, using a bio-computation based phosphorylation mapping method, the threonine 42 of CDB3 was identified to be the major target of casein kinase I. Finally, sequence analysis indicates that band 3 contains the conserved regions of an SH2 domain comprising cytoplasmically exposed loops of membrane-spanning domain separated by nonhomologous membrane-spanning sequences. The novel SH2 pocket may provide a docking site for the phosphorylated cytoplasmic domain and thus participates in the cell regulations