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Monoclonal Antibodies against Native and Denatured Forms of Estrogen-induced Breast Cancer Protein (BCEI/pS₂) Obtained by Expression in Escherichia coli¹

Groupe de Recherches sur la Biochimie Endocrinienne et la Reproduction (INSERM U. 135), Faculté de Médecine Paris-Sud, 94275 Le Kremlin-Bicêtre Cedex, France

Several vectors were used to express the complementary DNA for breast cancer estrogen-induced protein BCEI (also called pS₂) in Escherichia coli. The best results were obtained by using the pUR 290 expression vector after deletion of the sequence encoding the signal peptide of the protein. In these conditions, ß-galactosidase-BCEI/pS₂ fusion protein accounted for 20% of total proteins in bacterial extracts. It was purified by chromatography on DEAE-Trisacryl or by gel electrophoresis and electroelution.

Polyclonal antibodies were obtained by immunization of rabbits and goats, and monoclonal antibodies were raised in mice. Two types of monoclonal antibodies were obtained: one class recognized the native protein and was very efficient for the immunoprecipitation and immunopurification of the protein from breast cancer cells; a second class recognized the denatured protein and was especially effective for immunoblot studies.

BCEI/pS₂ could be detected by immunocytochemistry in breast cancer biopsies using monoclonal antibodies on frozen or paraffin-embedded sections. One of the antibodies (mBCEI₁₁) exhibited high affinity for the protein and could be used at 1.9 µg/ml concentration for immunolabeling of histological sections.

The mBCEI₁₁ antibody was used in immunoaffinity chromatography to purify the peptide in a single step from culture media of estrogen-treated MCF-7 cells.

¹ This work was supported by the Institut National de la Santé et de la Recherche Médicale, the Centre National de la Recherche Scientifique, the Unité d'Enseignement et de Recherches Kremlin-Bicêtre, the Association pour la Recherche sur le Cancer Transbio Company, and the Fondation pour la Recherche Médicale Française.

² To whom requests for reprints should be addressed.

Received 8/ 8/89. Revised 12/14/89.

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