CHARACTERIZATION OF RECOMBINANT E-CADHERIN (UVOMORULIN) EXPRESSED IN INSECT CELLS

Cadherins are Ca2+-dependent cell adhesion molecules that mediate cell adhesion by hemophilic binding. Structural and functional analysis of the extracellular part of cadherins that mediates this binding has of ten been hampered by the availability of sufficient amount of protein. Therefore, we have expressed the extracellular region of E-cadherin ( uvomorulin) using the baculovirus expression vector system (BEVS). A r ecombinant baculovirus was generated that encodes the signal peptide, the precursor region and the extracellular part of the mature protein, under the control of the promotor for polyhedrin. Infection of insect cells with recombinant virus led to the expression of about 40 mg of the E-cadherin fragment per 2x10(9) infected cells. About half of the protein synthesized was secreted, either as mature protein or in its u nprocessed form. The precursor peptide was removed by trypsin treatmen t in the presence of Ca2+ and recombinant protein was purified to homo geneity. Biochemical characterization of the recombinant protein revea led a high degree of similarity with the mouse wild-type protein. Reco mbinant protein exhibited the known resistance to trypsin in the prese nce of Ca2+ and was recognized by two different conformation-sensitive monoclonal anti-E-cadherin antibodies. Rabbit antibodies made against the recombinant protein recognized E-cadherin from different species. In spite of the high degree of structural resemblance recombinant E-c adherin was not able to inhibit E-cadherin mediated cell-cell adhesion .