Construction, expression, and purification of human recombinant tropomodulin and its binding to rTM5 and rTM5b.

(A) Human tropomodulin cDNA clone 10 was subcloned at the EcoR1 site downstream from the MalE gene, which encodes MBP. (B) The induction of the MBP/tropomodulin fusion protein. Total proteins ofE coli were separated on a 7% gel using SDS-PAGE and stained by Coomassie blue, before the addition of IPTG (lane 2) and 1 hour (lane 3), 2 hours (lane 4), and 3 hours (lane 5) after the addition of IPTG. Affinity-purified tropomodulin fusion protein (M_r 79 000) was either stained by Coomassie blue (lane 6, 2 μg) or transferred onto a nitrocellulose membrane and detected with tropomodulin-specific antibody using Western blot analysis (lane 7, 0.1 μg). Molecular weight standards (lane 1) are the same as in Figure 2. (C) A solid-phase binding assay demonstrates the binding of human tropomodulin to rTM5 and rTM5b. Using SDS-PAGE, 2 μg of rTM5 (lanes 1 and 3) and rTM5b (lanes 2 and 4) were separated on a 10% gel, transblotted onto a nitrocellulose membrane (panel labeled “Protein Profile”), and overlaid with 26 μg/mL recombinant human tropomodulin (panel labeled “Tmod Overlay”). The presence of bound tropomodulin was then analyzed by a rabbit antibody against tropomodulin followed by HRP-antirabbit IgG, enzymatic color development, and densitometry. (D) The panel shows the partial sequence alignments of human,16 mouse,32rat,33 and chicken38 tropomodulin in the region between residues 94 and 138. One letter code for amino acids is used. *Indicates identical residues among all 4 species. Numbers on the top indicate residue numbers in human tropomodulin.