title: Expression and purification of a soluble tissue factor fusion protein with an epitope for an unusual calcium-dependent antibody.
abstract: The use of bacterial signal peptides to target recombinant mammalian proteins to the periplasmic space of Escherichia coli (to promote proper disulfide bond formation) has met with variable success. We report the design and use of a bacterial expression vector to direct recombinant fusion proteins to the periplasmic space of E. coli: it contains the signal peptide from the pelB gene of Erwinia carotovora linked to a small peptide epitope for an unusual calcium-dependent antibody (HPC4). HPC4 binds to the epitope in a Ca(2+)-dependent manner, but the epitope itself does not bind Ca2+. We have used this system to express a biologically active, soluble form of tissue factor, the protein responsible for triggering the blood clotting cascade. Soluble tissue factor was secreted into the culture medium at 1-2 mg/liter, from which it could be readily purified using immobilized HPC4 antibody. The HPC4 epitope could be removed by digestion with thrombin or factor Xa, although a free amino terminus was not required for function since soluble tissue factor was equally active with the epitope still in place. This vector/epitope system permits large-scale expression and purification of recombinant soluble tissue factor and should be generally applicable to the isolation of other recombinant proteins. Furthermore, the epitope confers Ca(2+)-dependent binding of the fusion protein to HPC4 antibody while avoiding the creation of a new metal binding site on the fusion protein itself. Tb3+ can bind in this Ca2+ site near Trp, allowing this site to serve as a means of attaching a fluorescent probe to tissue factor.
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D000906:Antibodies / Q000187:drug effects; D000937:Antigen-Antibody Reactions; D001483:Base Sequence; D002118:Calcium / Q000494:pharmacology; D002451:Cell Compartmentation; D000939:Epitopes / Q000096:biosynthesis / Q000235:genetics; D005822:Genetic Vectors; D008969:Molecular Sequence Data; D011486:Protein C / Q000096:biosynthesis* / Q000235:genetics / Q000276:immunology; D021382:Protein Sorting Signals / Q000096:biosynthesis / Q000235:genetics; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000187:drug effects / Q000302:isolation & purification; D012995:Solubility; D013925:Thromboplastin / Q000096:biosynthesis* / Q000187:drug effects / Q000235:genetics
chemical_list: D000906:Antibodies; D000939:Epitopes; D011486:Protein C; D021382:Protein Sorting Signals; D011993:Recombinant Fusion Proteins; D013925:Thromboplastin; D002118:Calcium