Rapid Generation of Stable Cell Lines Expressing Corticotropin-Releasing Hormone Receptor for Drug Discovery

• Published in: Protein expression and purification Vol. 9; no. 3; pp. 301 - 308
• Main Authors: Horlick, Robert A., Sperle, Karen, Breth, Leah A., Reid, Christian C., Shen, Emily S., Robbins, Alan K., Cooke, Gary M., Largent, Brian L.
• Format: Journal Article
• Language: English
• Published: United States Elsevier Inc 01.04.1997
• Subjects: Animals; Cell Line; CHO Cells; Cricetinae; Drug Design; Epstein-Barr Virus Nuclear Antigens - genetics; Gene Expression; Genetic Vectors; Herpesvirus 4, Human - genetics; Humans; Plasmids - genetics; Receptors, Corticotropin-Releasing Hormone - biosynthesis; Receptors, Corticotropin-Releasing Hormone - classification; Receptors, Corticotropin-Releasing Hormone - genetics; Replication Origin; RNA - genetics; RNA - metabolism
• ISSN: 1046-5928; 1096-0279
• DOI: 10.1006/prep.1996.0701

Human HEK293 cells that stably express the Epstein Barr nuclear antigen 1 (EBNA1) support the episomal replication of plasmids containing the Epstein Barr virus origin of replication (EBV oriP). A 293EBNA (293E) cell line expressing the human corticotropin-releasing hormone receptor subtype I (CRHR1) from an episomal plasmid was generated (293CR1s), analyzed, adapted to spinner culture, and scaled-up for production in less than 6 weeks. Forty-seven stable CHO cell lines transfected with CRHR1 were also isolated. Expression of the receptor in the best of these lines (as judged by CRH-induced cAMP production), CHO-R22, was compared to that in 293CR1s cells. Results indicate that the CRHR1 episomal expression vector in 293E cells (1) rapidly generates stable cell lines suitable for scale-up; (2) is stably maintained during 3 months in culture; (3) expresses high levels of CRHR1 mRNA; and (4) expresses significantly more CRHR1 than the CHO-R22 line. Coexpression of additional G protein α subunit (Gαs) with CRHR1 in 293E cells converts a higher percentage of receptor to the agonist high-affinity G-protein-coupled state. Our data support the idea that using the EBV oriP-driven episomal system for gene expression results in greater production of protein in a relatively short period of time.