zWe expressed gram amounts of full-length and mutant rat brain calcium-binding proteins (calbindins-D28K) lacking one or two "EF-hand" motifs in a bacterial expression system. The cDNA for the full-length rat calcium-binding protein was cloned into the NdeI and BamHI sites of the pET3a vector. Additionally, constructs of the rat brain calcium-binding protein lacking EF-hand 2 (Δ2 mutant), EF-hand 6 (Δ6 mutant), and EF-hands 2 and 6 (Δ2,6 mutant) were constructed using the same vector. These chimeric plasmids were used to transfect BL21(DE3) pLysS Escherichia coli cells. Following transformation, the cells were grown in the presence of isopropylthiogalactoside in order to induce bacterial T7 polymerase, which resulted in the production of large amounts of the proteins of interest in the bacterial cytosol. Expressed full-length and Δ2 and Δ2,6 mutant proteins represented 50% or more of total bacterial protein. The Δ6 protein was not expressed. Cell lysis followed by purification of the proteins on DEAE-cellulose routinely resulted in gram yields of the proteins. The purified proteins displayed the appropriate amino acid composition and amino-terminal amino acid sequence. When analyzed by matrix-assisted laser desorption mass spectrometry the proteins were found to have the appropriate molecular weights (within the accuracy limits of the instrument). The expressed proteins bound to a polyclonal antiserum raised against chick intestinal calcium-binding protein. In addition, the full-length, Δ2, and Δ2,6 mutants bound calcium as assessed by a 45Ca blotting procedure. The production of large amounts of readily purified vitamin D-dependent calcium-binding proteins should be useful in biophysical studies of the proteins.
ASJC Scopus subject areas
- Molecular Biology