Characterization of recombinant E-cadherin (uvomorulin) expressed in insect cells.
Cadherins are Ca(2+)-dependent cell adhesion molecules that mediate cell adhesion by homophilic binding. Structural and functional analysis of the extracellular part of cadherins that mediates this binding has often 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 recombinant 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 2 x 10(9) infected cells. About half of the protein synthesized was secreted, either as mature protein or in its unprocessed form. The precursor peptide was removed by trypsin treatment in the presence of Ca2+ and recombinant protein was purified to homogeneity. Biochemical characterization of the recombinant protein revealed a high degree of similarity with the mouse wild-type protein. Recombinant protein exhibited the known resistance to trypsin in the presence 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-cadherin was not able to inhibit E-cadherin mediated cell-cell adhesion.
Herrenknecht
K
K
Max-Planck-Institut für Immunbiologie, Freiburg, Germany.
Kemler
R
R
D000818:Animals; D000906:Antibodies; D015820:Cadherins / Q000235:genetics / Q000276:immunology / Q000378:metabolism*; D002240:Carbohydrate Sequence; D002448:Cell Adhesion; D002460:Cell Line; D015870:Gene Expression; D005822:Genetic Vectors; D006031:Glycosylation; D007120:Immunochemistry; D008969:Molecular Sequence Data; D009036:Moths; D017924:Nucleopolyhedroviruses / Q000235:genetics; D011994:Recombinant Proteins / Q000235:genetics / Q000276:immunology / Q000378:metabolism
D000906:Antibodies; D015820:Cadherins; D011994:Recombinant Proteins